Emulsion vehicle for poorly soluble drugs

ABSTRACT

A method of making an emulsion of tocopherol incorporating a co-solvent and, stabilized by biocompatible surfactants, as a vehicle or carrier for therapeutic drugs, which is substantially ethanol free and which can be administered to animals or humans by various routes is disclosed. Also included in the emulsion is PEGylated vitamin E. PEGylated α-tocopherol includes polyethylene glycol subunits attached by a succinic acid diester at the ring hydroxyl of vitamin E and serves as a primary surfactant, stabilizer and a secondary solvent in emulsions of α-tocopherol.

RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) ofU.S. provisional application No. 60/088,269 filed Jun. 5, 1998.

FIELD OF THE INVENTION

[0002] This invention is in the field of pharmaceutical agents. Inparticular, this invention relates to pharmaceutical agents whereintocopherol is used as a primary solvent.

BACKGROUND OF THE INVENTION

[0003] Hundreds of medically useful compounds are discovered each year,but clinical use of these drugs is possible only if a drug deliveryvehicle is developed to transport them to their therapeutic target inthe human body. This problem is particularly critical for drugsrequiring intravenous injection in order to reach their therapeutictarget or dosage but which are water insoluble or poorly water soluble.For such hydrophobic compounds, direct injection may be impossible orhighly dangerous, and can result in hemolysis, phlebitis,hypersensitivity, organ failure and/or death. Such compounds are termedby pharmacists “lipophilic”, “hydrophobic”, or in their most difficultform, “amphiphobic”.

[0004] A few examples of therapeutic substances in these categories areibuprofen, diazepam, griseofulvin, cyclosporin, cortisone, proleukin,etoposide and paclitaxel. Kagkadis, K A et al. (1996) PDA J Pharm SciTech 50(5):317-323; Dardel, O. 1976. Anaesth Scand 20:221-24. Sweetana,S and M J U Akers. (1996) PDA J Pharm Sci Tech 50(5):330-342.

[0005] Administration of chemotherapeutic or anti-cancer agents isparticularly problematic. The majority of these agents are poorlysoluble and thus are difficult to deliver in aqueous solvents and supplyat therapeutically useful levels. On the other hand, water-solubleanti-cancer agents are generally taken up by both cancer and non-cancercells thereby exhibiting non-specificity.

[0006] Efforts to improve water-solubility and comfort of administrationof such agents have not solved, and may have worsened, the twofundamental problems of cancer chemotherapy: 1) non-specific toxicityand 2) rapid clearance form the bloodstream by non-specific mechanisms.In the case of cytotoxins, which form the majority of currentlyavailable chemotherapies, these two problems are clearly related.Whenever the therapeutic is taken up by non-cancerous cells, adiminished amount of the drug remains available to treat the cancer, andmore importantly, the normal cell ingesting the drug is killed.

[0007] To be effective in treating cancer, the chemotherapeutic must bepresent throughout the affected tissue(s) at high concentration for asustained period of time so that it may be taken up by the cancer cells,but not at so high a concentration that normal cells are injured beyondrepair. Obviously, water soluble molecules can be administered in thisway, but only by slow, continuous infusion and monitoring, aspects whichentail great difficulty, expense and inconvenience.

[0008] A more effective method of administering a cancer therapeutic,particularly a cytotoxin, is in the form of a dispersion of oil in whichthe drug is dissolved. These oily particles are made electricallyneutral and coated in such a way that they do not interact with plasmaproteins and are not trapped by the reticuloendothelial system (RES),instead remaining intact in the tissue or blood for hours, days or evenweeks. It is desirable when the particles also distribute themselvesinto the surrounding lymph nodes which are injected at the site of acancer. Nakamoto, Y et al. (1975) Chem Pharm Bull 23(10):2232-2238.Takahashi, T et al. (1977) Tohoku J Exp Med 123:235-246. In many casesdirect injection into blood is the route of choice for administration.Even more preferable, following intravenous injection, the blood-borneparticles may be preferentially captured and ingested by the cancercells themselves. An added advantage of a particulate emulsion for thedelivery of a chemotherapeutic is the widespread property of surfactantsused in emulsions to overcome multidrug resistance.

[0009] For drugs that cannot be formulated as an aqueous solution,emulsions have typically been most cost-effective and gentle toadminister, although there have been serious problems with making themsterile and endotoxin free so that they may be administered byintravenous injection. The oils typically used for pharmaceuticalemulsions include saponifiable oils from the family of triglycerides,for example, soybean oil, sesame seed oil, cottonseed oil, safflower oiland the like. Hansrani, P K et al., (1983) J. Parenter Sci. Technol37:145-150. One or more surfactants are used to stabilize the emulsion,and excipients are added to render the emulsion more biocompatible,stable and less toxic. Lecithin from egg yolks or soybeans is a commonlyused surfactant. Sterile manufacturing can be accomplished by absolutesterilization of all the components before manufacture, followed byabsolutely aseptic technique in all stages of manufacture. However,improved ease of manufacture and assurance of sterility is obtained byterminal sterilization following sanitary manufacture, either by heat orby filtration. Unfortunately, not all emulsions are suitable for heat orfiltration treatments.

[0010] Stability has been shown to be influenced by the size andhomogeneity of the emulsion. The preferred emulsion consists of asuspension of sub-micron particles, with a mean droplet diameter of nogreater than 200 nanometers. A stable dispersion in this size range isnot easily achieved, but has the benefit that it is expected tocirculate longer in the bloodstream. Further, less of the stabledispersion in this size range is phagocytized non-specifically by thereticuloendothelial system. As a result the drug is more likely to reachits therapeutic target. Thus, a preferred drug emulsion will be designedto be actively taken up by the target cell or organ, and is targetedaway from the RES.

[0011] The use of vitamin E in emulsions is known. In addition to thehundreds of examples where vitamin E in small quantities (for example,less than 1%, Lyons, R. T., Pharm Res 13(9): S-226, (1996) “Formulationdevelopment of an injectable oil-in-water emulsion containing thelipophilic antioxidants α-tocopherol and β-carotene”) is used as ananti-oxidant in emulsions, the first primitive, injectable vitamin Eemulsions per se were made by Hidiroglou for dietary supplementation insheep and for research on the pharmacokinetics of vitamin E and itsderivatives. Hidiroglou M. and Karpinski K. (1988) Brit J Nutrit59:509-518.

[0012] For mice, an injectable form of vitamin E was prepared by Katoand coworkers. Kato Y., et al. (1993) Chem Pharm Bull 41(3):599-604.Micellar solutions were formulated with Tween 80, Brij 58 and HCO-60.Isopropanol was used as a co-solvent, and was then removed by vacuumevaporation; the residual oil glass was then taken up in water withvortexing as a micellar suspension. An emulsion was also prepared bydissolving vitamin E with soy phosphatidycholine (lecithin) and soybeanoil. Water was added and the emulsion prepared with sonication.

[0013] In 1983, E-Ferol, a vitamin E emulsion was introduced for vitaminE supplementation and therapy in neonates. Alade S. L. et al. (1986)Pediatrics 77(4):593-597. Within a few months over 30 babies had died asa result of receiving the product, and the product was promptlywithdrawn by FDA order. The surfactant mixture used in E-Ferol toemulsify 25 mg/mL vitamin E consisted of 9% Tween 80 and 1% Tween 20.These surfactants at the employed levels seem ultimately to-have beenresponsible for the unfortunate deaths. This experience illustrates theneed for improved formulations and the importance of selecting suitablebiocompatible surfactants and carefully monitoring their levels inparenteral emulsions.

[0014] An alternative means of solubilizing low solubility compounds isdirect solubilization in a non-aqueous milieu, for example alcohol (suchas ethanol) dimethylsulfoxide or triacetin. An example in PCTapplication WO 95/11039 describes the use of vitamin E and the vitamin Ederivative TPGS in combination with ethanol and the immuno-suppressantmolecule cyclosporin. U.S. Pat. No. 5,689,846 discloses various alcoholsolutions of paclitaxel. U.S. Pat. No. 5,573,781 discloses thedissolution of paclitaxel in ethanol, butanol and hexanol and anincrease in the antitumor activity of paclitaxel when delivered inbutanol and hexanol as compared to ethanol. Alcohol-containing solutionscan be administered with care, but are typically given by intravenousdrip to avoid the pain, vascular irritation and toxicity associated withbolus injection of these solutions.

[0015] PCT publication WO 95/21217 (Dumex Ltd) discloses thattocopherols can be used as solvents and/or emulsifiers of drugs that aresubstantially insoluble in water, in particular for the preparation oftopical formulations. The use of vitamin E-TPGS as an emulsifier informulations containing high levels of α-tocopherol is mentioned in thespecification (pages 7-8 and 12). Examples 1 to 5, disclosedformulations for topical administration comprising a lipid layer(α-tocopherol), the drug and Vitamin E-TPGS, in quantities of less than25% w/w of the formulation, as an emulsifier. WO95/21217 does notsuggest or describe anticancer agents or taxanes.

[0016] PCT Publication WO 97/03651 (Danbiosyst UK Ltd.) discloses lipidvehicle drug delivery compositions that contain at least fiveingredients: a therapeutic drug, vitamin E, an oil in which the drug andvitamin E are dissolved, a stabilizer (either phospholipid, a lecithin,or a poloxamer which is a polyoxyethylene-polyoxypropylene copolymer)and water. The therapeutic drugs disclosed are itraconazole andpaclitaxel. The “therapeutic emulsion” compositions require two oils inthe dispersed phase where the therapeutic drug resides, vitamin E andanother oil, typically a triglyceride such as soybean oil. The onlyworking example with paclitaxel, Example 16, also contains both vitaminE and soybean oil.

[0017] N-methyl-2-pyrrolidone (NMP), under the trade name Pharmosolve™,can be used to improve the solubility of poorly soluble drugs inpharmaceutical formulations and has appeared in recent literature foruse in veterinary medicine with forthcoming application in humans.Furthermore, polyvinylpyrrolidone (PVP) under the trade name Povidone™with a molecular weight between 2,500 to 100,000 at a concentration of 1to 5 percent (w/v) of the aqueous injectable base can be used as aco-solubilizer along with NMP. U.S. Pat. No. 5,726,181 disclosesantitumor compositions and suspensions comprising NMP and highlylipophilic camptothecin derivatives.

[0018] Polyethylene glycols (PEGs) and PVP are examples of twowater-soluble polymers frequently used to modify the solubility behaviorof drugs, including paclitaxel. Although the solubility of paclitaxel inboth solvents is relatively high, in dilute aqueous solutions that aresuitable for parenteral administration the solubility of the drug is lowand the potential for drug precipitation upon dilution is high. Inadmixtures of PEG 400 and water containing 50-100% PEG 400, thesolubility of paclitaxel varies from 0.2 to 175 mg/ml, respectively.Thus, paclitaxel solubilities are quite low where larger amounts ofwater are used, e.g., in 35% PEG 400 and 30% PVP in water are 0.03 mg/mland ≦0.3 mg/ml, respectively. “Solubility of paclitaxel in PolyethyleneGlycol 400/Water Mixtures” (Sraubinger, R. M. Biopharmacuitics ofpaclitaxel (Taxol); Formulation, activity and pharacokinetics, p.244 InTaxol, Science and Applications. (M. Suffness ed.), CRC Press, New York,1995). The use of PEG-400 is not limited to paclitaxel and can beapplied to other therapeutic agents which exhibit good solubility inpolyethylene glycols (for example Etoposide). Derivative forms ofpaclitaxel including polyethylene glycol derivatives are described inU.S. Pat. No. 5,614,549.

[0019] In addition to poor solubility and the potential for drugprecipitation with pharmaceutical formulations in non-aqueous solventssuch as alcohol (ethanol, isopropanol, benzyl alcohol, etc.) along withsurfactants another problem is the ability of these solvents to extracttoxic substances, for example plasticizers, from their containers. Thecurrent commercial formulation for the anti-cancer drug paclitaxel, forexample, consists of a mixture of hydroxylated castor oil and ethanol,and rapidly extracts plasticizers such as di-(2-ethylhexyl)-phthalatefrom commonly used intravenous infusion tubing and bags. Adversereactions to the plasticizers have been reported, such as respiratorydistress, necessitating the use of special infusion systems at extraexpense and time. Waugh, et al. (1991) Am J. Hosp. Pharmacists 48:1520.

[0020] In light of these problems, it can be seen that the idealemulsion vehicle would be inexpensive, non-irritating or even nutritiveand palliative in itself, terminally sterilizable by either heat orfiltration, stable for at least 1 year under controlled storageconditions, accommodate a wide variety of water insoluble and poorlysoluble drugs and be substantially ethanol-free. In addition to thosedrugs which are lipophilic and dissolve in oils, also needed is avehicle which will stabilize, and carry in the form of an emulsion,drugs which are poorly soluble in lipids and in water.

SUMMARY OF THE INVENTION

[0021] In order to meet these needs, the present invention is directedto pharmaceutical compositions including: tocopherol, with and withoutan aqueous phase, a surfactant or mixtures of surfactants incorporatinga co-solvent and a therapeutic agent. The compositions of the inventionmay be in the form of an emulsion, micellar solution or aself-emulsifying drug delivery system. The tocopherol molecule ispreferably α-tocopherol. The compositions of the invention are generallysubstantially free of any monohydric alcohol.

[0022] The co-solvent may include water-soluble polymers, preferablypolyethylene glycols or polyvinylpyrrolidone with or withoutN-methyl-2-pyrrolidone. Polyethylene glycols (PEGs) with a molecularweight between 100 to 10,000 are the most preferred co-solvent. Mostpreferred is PEG-400 in amounts greater than 1% by weight of theformulation.

[0023] The pharmaceutical compositions can be stabilized by the additionof various amphiphilic molecules, including anionic, nonionic, cationic,and zwitterionic surfactants. Preferably, these molecules are PEGylatedsurfactants and optimally PEGylated α-tocopherol.

[0024] The amphiphilic molecules further include surfactants such asascorbyl-6 palmitate; stearylamine; sucrose fatty acid esters, pegylatedphospholipids, various vitamin E derivatives and fluorine-containingsurfactants (such as the Zonyl brand series) and apolyoxypropylene-polyoxyethylene glycol nonionic block copolymer.

[0025] The therapeutic agent of the emulsion may be a chemotherapeuticagent preferably a taxoid analog and most preferably, paclitaxel.

[0026] The emulsions of the invention can comprise an aqueous mediumwhen in the form of an emulsion or micellar solution. This medium cancontain various additives to assist in stabilizing the emulsion or inrendering the formulation biocompatible.

[0027] In one form, the invention is directed to a pharmaceuticalcomposition comprising α-tocopherol, a chemotherapeutic selected fromtaxoids, taxins and taxanes, water and D-α-tocopherol polyethyleneglycol1000 succinate. In another form, the invention is directed to apharmaceutic composition comprising α-tocopherol, a co-solvent, one ormore surfactants, an aqueous phase and a therapeutic agent wherein thecomposition is in the form of an emulsion or micellar solution and thesolution is substantially free of any monohydric alcohol.

[0028] In a preferred format, the co-solvent may be polyethylene glycol,N-methyl-2-pyrrolidone, polyvinyl-pyrrolidone or mixtures thereof.

[0029] In a preferred format the surfactant is an α-tocopherolderivative and the polyethylene glycol has a molecular weight between100 to 10,000 most preferably from about 200 to about 1000.

[0030] In a preferred format the therapeutic agent is a chemotherapeuticagent selected from taxoids, taxines and taxanes.

[0031] The pharmaceutical compositions of the invention are typicallyformed by dissolving a therapeutic agent in the co-solvent to form atherapeutic agent solution; α-tocopherol is then added along with one ormore surfactants to the therapeutic agent solution to form an oilsolution of the therapeutic agent in the hydrophilic co-solvent. The oilsolution is then blended with an aqueous phase to form a pre-emulsion.For IV delivery the pre-emulsion is further homogenized to form a fineemulsion. For oral delivery, the oil solution of the therapeutic agentin the co-solvent along with surfactants is typically encapsulated in agelatin capsule.

[0032] In a preferred form of the method of the invention thetherapeutic agent is dissolved in polyethylene glycol which allows theavoidance of the use of monohydric alcohols as a solvent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The invention will be better understood by reference to thefigures, in which:

[0034]FIG. 1A shows the particle size of a paclitaxel emulsion (QWA) at7° C. over time;

[0035]FIG. 1B shows the particle size of a paclitaxel emulsion (QWA) at25° C. over time;

[0036]FIG. 2 is an HPLC chromatogram showing the integrity of apaclitaxel in an emulsion as described in Example 5;

[0037]FIG. 3A shows the paclitaxel concentration of a paclitaxelemulsion (QWA) at 4° C. over time;

[0038]FIG. 3B shows the paclitaxel concentration of a paclitaxelemulsion (QWA) at 25° C. over time; and

[0039]FIG. 4 shows the percentage of paclitaxel released over time fromthree different emulsions. The symbol  represents the percentage ofpaclitaxel released over time from an emulsion commercially availablefrom Bristol Myers Squibb. The symbol □ represents the percentage ofpaclitaxel released over time from an emulsion of this inventioncontaining 6 mg/ml paclitaxel (QWA) as described in Example 6. Thesymbol represents the percentage of paclitaxel released over time froman emulsion of this invention (QWB) containing 7 mg/ml paclitaxel asdescribed in Example 7.

[0040]FIG. 5 shows the efficacy of a PEG-400/Vitamin E/paclitaxelemulsion against B16 melanoma in nice.

DETAILED DESCRIPTION OF THE INVENTION

[0041] To ensure a complete understanding of the invention the followingdefinitions are provided:

[0042] Tocopherols: tocopherols are a family of natural and syntheticcompounds, also known by the generic names tocols or Vitamin E.α-tocopherol, is the most abundant and active form of this class ofcompounds and it has the following chemical structure (Scheme I):

[0043] Other members of this class include α-, β-, γ-, andδ-tocotrienols, and α-tocopherol derivatives such as tocopherol acetate,phosphate, succinate, nitotinate and linoleate. In addition to their useas a primary solvent, tocopherols and their derivatives are useful as atherapeutic agents.

[0044] Surfactants: Surface active group of amphiphilic molecules whichare manufactured by chemical processes or purified from natural sourcesor processes. These can be anionic, cationic, nonionic, andzwitterionic. Typical surfactants are described in Emulsions: Theory andPractice, Paul Becher, Robert E. Krieger Publishing, Malabar, Fla.,1965; Pharmaceutical Dosage Forms: Dispersed Systems Vol. 1, Martin M.Rigear, Surfactants and U.S. Pat. No. 5,595,723 which is assigned to theassignee of this invention, Sonus Pharmaceuticals. All of thesereferences are hereby incorporated by reference.

[0045] TPGS: TPGS or PEGylated vitamin E is a vitamin E derivative inwhich polyethylene glycol subunits are attached by a succinic aciddiester at the ring hydroxyl of the vitamin E molecule. TPGS stands forD-α-tocopherol polyethyleneglycol 1000 succinate (MW=1513). TPGS is anon-ionic surfactant (HLB=16-18) with the structure of Scheme II:

[0046] Various chemical derivatives of vitamin E TPGS including esterand ether linkages of various chemical moieties are included within thedefinition of vitamin E TPGS.

[0047] Polyethylene glycol: Polyethylene glycol (PEG) is a hydrophilic,polymerized form of ethylene glycol, consisting of repeating units ofthe chemical structure—(CH₂—CH₂—O—). The general formula forpolyethylene glycol is HOCH₂ (CH₂OCH₂)_(n) CH₂OH or H(OCH₂CH₂)_(n)OH.The molecular weight ranges from 200 to 10,000. Such various forms aredescribed as PEG-200, PEG-400 and the like.

[0048] N-Methyl-2-pyrrolidone: N-methyl-2-pyrrolidone (NMP) is anorganic molecule with the following chemical structure:

[0049] A GMP grade of this compound is available under the namePharmasolve™ and is used to improve the solubility of poorly solubledrugs in pharmaceutical formulations. The enhanced solubility of certaindrugs can be attributed to a complexing action with the nitrogen andcarbonyl reactive centers of the molecule.

[0050] Polyvinyl pyrrolidone: Polyvinyl pyrrolidone (PVP) or Povidone isa water soluble polymer, consisting of repeatings units of the chemicalstructure:

[0051] It's average MW can vary between 2500 and 3×10⁶ special grades ofpyrogen free povidone are available for parenteral administration.Concentrations up to 5% w/v can be used as co-solvent for poorly solubledrugs.

[0052] Poloxamers or Pluronics: are synthetic block copolymers ofethylene oxide and propylene oxide having the general structure:

OH (OCH₂CH₂)a (OCH₂CH₂CH₂)b (OCH₂CH₂)a H

[0053] The following variants based on the values of a and b arecommercially available from BASF Performance Chemicals (Parsippany,N.J.) under the trade name Pluronic and which consist of the group ofsurfactants designated by the CTFA name of Poloxamer 108, 188, 217, 237,238, 288, 338, 407, 101, 105, 122, 123, 124, 181, 182, 183, 184, 212,231, 282, 331, 401, 402, 185, 215, 234, 235, 284, 333, 334, 335, and403. For the most commonly used poloxamers 124, 188, 237, 338 and 407the values of a and b are 12/20, 79/28, 64/37, 141/44 and 101/56,respectively.

[0054] Solutol HS-15: is a polyethylene glycol 660 hydroxystearatemanufactured by BASF (Parsippany, N.J.). Apart from free polyethyleneglycol and its monoesters, diesters are also detectable. According tothe manufacturer, a typical lot of Solutol HS-15 contains approximately30% free polyethylene glycol and 70% polyethylene glycol esters.

[0055] Other surfactants: Other surfactants useful in the inventioninclude ascorbyl-6 palmitate (Roche Vitamins, Nutley, N.J.),stearylamine, and sucrose fatty acid esters (Mitsubishi Chemicals).Custom surfactants include those compounds with polar water-loving headsand hydrophobic tails, such as a vitamin E derivative comprising apeptide bonded polyglutamate attached to the ring hydroxyl and pegylatedphytosterol. Other peptides may be bonded to vitamin E as well. Alsopegylated phospholipids are useful surfactants. Examples of pegylatedphospholipids include PEG 2000 or PEG 5000 analogs ofphosphatidylethanolamine where the fatty acyl chains contain C₆-C₂₄fatty acids which can be saturated, unsaturated, mixtures thereof.

[0056] Hydrophile-lipophile balance: An empirical formula used to indexsurfactants. Its value varies from 1-45 and in the case of non-ionicsurfactants from about 1-20. In general for lipophilic surfactants theHLB is less than 10 and for hydrophilic ones the HLB is greater than 10.

[0057] Biocompatible: Capable of performing functions within or upon aliving organism in an acceptable manner, without undue toxicity orphysiological or pharmacological effects.

[0058] Substantially free of any monohydric alcohol: A compositionhaving a monohydric alcohol concentration less than about 1.0% (w/v)monohydric alcohol. As used herein, the term “monohydric” alcohol is analcohol containing one hydroxyl group, such as but not limited toethanol, butanol, isopropanol. The term “polyhydric” alcohol or “polyol”is an alcohol containing two or more hydroxyl groups, such as but notlimited to, ethylene glycol, propylene glycol or polyethylene glycol(PEG). PEG is also referred to as polyglycol with ethylene glycol as apolymerized unit. Other suitable polyhydric alcohols for use hereininclude, but are not limited to, ethylene glycol (2-OH groups), glycerol(3-OH groups), sorbitol (6-OH groups) and mannitol (6-OH groups).

[0059] Emulsion: A colloidal dispersion of two immiscible liquids in theform of droplets, whose diameter, in general, are between 0.1 and 3.0microns and which is typically optically opaque, unless the dispersedand continuous phases are refractive index matched. Such systems possessa finite stability, generally defined by the application or relevantreference system, which may be enhanced by the addition of amphiphilicmolecules or viscosity enhancers.

[0060] Microemulsion: A thermodynamically stable isotropically cleardispersion of two immiscible liquids, such as oil and water, stabilizedby an interfacial film of surfactant molecules. The microemulsion has amean droplet diameter of less than 200 nm, in general between 10-50 nm.In the absence of water, mixtures of oil(s) and non-ionic surfactant(s)form clear and isotropic solutions that are known as self-emulsifyingdrug delivery systems (SEDDS) and have successfully been used to improvelipophilic drug dissolution and oral absorption.

[0061] Pegylated: Pegylated or ethoxylated means polyethylene glycolsubunits attached to a given compound via a chemical linkage.

[0062] Aqueous Medium: A water-containing liquid which can containpharmaceutically acceptable additives such as acidifying, alkalizing,buffering, chelating, complexing and solubilizing agents, antioxidantsand antimicrobial preservatives, humectants, suspending and/or viscositymodifying agents, tonicity and wetting or other biocompatible materials.

[0063] Therapeutic Agent: Any compound natural of synthetic which has abiological activity, is soluble in the oil phase and has anoctanol-buffer partition coefficient (Log P) of at least 2 to ensurethat the therapeutic agent is preferentially dissolved in the oil phaserather than the aqueous phase. This includes peptides, non-peptides andnucleotides. Hydrophobic derivatives of water soluble molecules such aslipid conjugates/prodrugs are within the scope of therapeutic agent.

[0064] Chemotherapeutic: Any natural or synthetic molecule which iseffective against one or more forms of cancer, and particularly thosemolecules which are slightly or completely lipophilic or which can bemodified to be lipophilic. This definition includes molecules which bytheir mechanism of action are cytotoxic (anti-cancer agents), thosewhich stimulate the immune system (immune stimulators) and modulators ofangiogenesis. The outcome in either case is the slowing of the growth ofcancer cells.

[0065] Chemotherapeutics include Taxol (paclitaxel) and relatedmolecules collectively termed taxoids, taxines or taxanes. The structureof paclitaxel is shown in the figure below (Scheme V).

[0066] Included within the definition of “taxoids”are variousmodifications and attachments to the basic ring structure (taxoidnucleus) as may be shown to be efficacious for reducing cancer cellgrowth and to partition into the oil (lipid phase) and which can beconstructed by organic chemical techniques known to those skilled in theart. These include but are not limited to benzoate derivatives ofpaclitaxel such as 2-debenzoyl-2-aroyl and C-2-acetoxy-C-4-benzoatepaclitaxel, 7-deocytaxol, C-4 aziridine paclitaxel, as wells as variouspaclitaxel conjugates with natural and synthetic polymers, particularlywith fatty acids, phospholipids, and glycerides and1,2-diacyloxypropane-3-amine. Docetaxel (Taxotere) is also a preferredtaxane. The structure of the taxoid nucleus is shown in Scheme VI.

[0067] Also included within the scope of the present invention arenatural products that share structural similarities with paclitaxel i.e.they incorporate a common pharmacophore proposed formicrotubule-stabilizing agents. These compounds include but not limitedto epothilone A and B, discodermolide, nonataxel and eleutherobin (Chem.Eng. News 1999, 77 (17): 35-36)

[0068] Chemotherapeutics include podophyllotoxins and their derivativesand analogues. The core ring structure of these molecules is shown inthe following figure (Scheme VII):

[0069] Another important class of chemotherapeutics useful in thisinvention are camptothecins, the basic ring structure of which is shownin the following figure, but includes any derivatives and modificationsto this basic structure which retain efficacy and preserve thelipophilic character of the molecule shown below (Scheme VIII).

[0070] Another preferred class of chemotherapeutics useful in thisinvention are the lipophilic anthracyclines, the basic ring structure ofwhich is shown in the following figure (Scheme IX):

[0071] Suitable lipophilic modifications of Scheme IX includesubstitutions at the ring hydroxyl group or sugar amino group.

[0072] Another important class of chemotherapeutics are compounds whichare lipophilic or can be made lipophilic by molecular chemosyntheticmodifications well known to those skilled in the art, for example bycombinatorial chemistry and by molecular modelling, and are drawn fromthe following list: Taxotere, Amonafide, Illudin S,6-hydroxymethylacylfulvene Bryostatin 1, 26-succinylbryostatin 1,Palmitoyl Rhizoxin, DUP 941, Mitomycin B, Mitomycin C, Penclomedine.Interferon α2b, angiogenesis inhibitor compounds, Cisplatin hydrophobiccomplexes such as 2-hydrazino-4,5-dihydro-1H-imidazole with platinumchloride and 5-hydrazino-3,4-dihydro-2H-pyrrole with platinum chloride,vitamin A, vitamin E and its derivatives, particularly tocopherolsuccinate.

[0073] Other compounds useful in the invention include:1,3-bis(2-chloroethyl)-1-nitrosurea (“carmustine” or “BCNU”),5-fluorouracil, doxorubicin (“adriamycin”), epirubicin, aclarubicin,Bisantrene(bis(2-imidazolen-2-ylhydrazone)-9,10-anthracenedicarboxaldehyde,mitoxantrone, methotrexate, edatrexate, muramyl tripeptide, muramyldipeptide, lipopolysaccharides, 9-b-d-arabinofuranosyladenine(“vidarabine”) and its 2-fluoro derivative, resveratrol, retinoic acidand retinol, Carotenoids, and tamoxifen.

[0074] Other compounds useful in the application of this inventioninclude: Decarbazine, Lonidamine, Piroxantrone, Anthrapyrazoles,Etoposide, Camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin,camptothecin-11 (“Irinotecan’), Topotecan, Bleomycin, the Vincaalkaloids and their analogs [Vincristine, Vinorelbine, Vindesine,Vintripol, Vinxaltine, Ancitabine], 6-aminochrysene, and navelbine.

[0075] Other compounds useful in the application of the invention aremimetics of taxol, eleutherobins, sarcodictyins, discodermolides andepothiolones.

[0076] Other compounds useful in the invention are microtubule targetingagents. Microtubule targeting agents may bind to a protein calledtubulin and thus prevent microtubule polymerization. Representativemicrotubule binding agents include epothilones, elutherobin anddiscodermolide.

[0077] Taking into account these definitions, the present invention isdirected to pharmaceutical compositions in the form of emulsions,micellar solutions or self-emulsifying drug delivery systems which aresubstantially free of ethanol solvent.

[0078] The therapeutic agents of the compositions of this invention caninitially be solubilized in a co-solvent. In the case of ethanol duringthe preparation of the oil phase the ethanol is removed and asubstantially ethanol-free composition is formed. The ethanolconcentration is less than 1% (w/v), preferably less than 0.5%, and mostpreferably less than 0.3%. The therapeutic agents can also besolubilized in methanol, propanol, chloroform, isopropanol, butanol andpentanol. These solvents are also removed prior to use.

[0079] In a preferred embodiment, the therapeutic agents of thecompositions of the invention can initially be solubilized innon-volatile co-solvents such as dimethylsulfoxide (DMSO), dimethylamide(DMA), propylene glycol (PG), polyethylene glycol (PEG),N-methyl-2-pyrrolidone (NMP) and polyvinylpyrrolidone (PVP); NMP or awater-soluble polymer such as PEG or PVP (Table 1) are particularlypreferred.

[0080] A major advantage/improvement of using PEG-400 to solubilizetherapeutic agents rather than alcohols such as ethanol is that avolatile solvent does not have to be removed or diluted prior toadministration of the therapeutic agent. The final polyethylene glycollevels in the emulsion can be varied from 1-50%, preferably from 1-25%and more preferably from 1-10% (w/w). Suitable polyethylene glycolsolvents are those with an average molecular weight between 200 and 600preferably between 300 and 400 (Table 1). In the case of self-emulsifiedsystems for oral administration, high molecular weight PEGs(1,000-10,000) can also be included as solidification agents to formsemi-solid formulations which can be filled into hard gelatin capsules.TABLE 1 Physical Properties of Low Molecular Weight Polyethylene GlycolsPhysical Property PEG 200 PEG 300 PEG 400 PEG 600 Molecular Weight190-210 285-315 380-420 570-630 Viscosity (mPas) 46-53 66-74 85-95130-150 Refractive Index 1.459 1.463 1.465 1.467 (25° C.) Freezing point(° C.) −50 −16 to −12 −3 to 8 15 to 25

[0081] Solubilization of the therapeutic agents of the invention inpolyethylene glycol or other non-volatile co-solvents (PVP, NMP) avoidsthe necessity of solubilizing the therapeutic agents of the invention inmonohydric alcohols such as ethanol or other volatile solvents. Use ofpolyethylene glycol or N-methyl-2-pyrrolidone eliminates the need toremove the solvent prior to use of the emulsions therapeutically.

[0082] The final polyethylene glycol levels in the emulsion can bevaried from 1-50%, preferably from 1-25% and more preferably from 1-10%(w/w).

[0083] The compositions of the invention contain tocopherol as a carrierfor therapeutic drugs, which can be administered to animals or humansvia intravascular, oral, intramuscular, cutaneous and subcutaneousroutes. Specifically, the emulsions can be given by any of the followingroutes, among others: intraabdominal, intraarterial, intraarticular,intracapsular, intracervical, intracranial, intraductal, intradural,intralesional, intralocular, intralumbar, intramural, intraocular,intraoperative, intraparietal, intraperitoneal, intrapleural,intrapulmonary, intraspinal, intrathoracic, intratracheal,intratympanic, intrauterine, and intraventricular. The emulsions of thepresent invention can be nebulized using suitable aerosol propellantswhich are known in the art for pulmonary delivery of lipophiliccompounds.

[0084] In its first aspect, the invention is directed to the use oftocopherol as the hydrophobic dispersed phase of emulsions containingwater insoluble, poorly water soluble therapeutic agents, water solubleones which have been modified to be less water soluble or mixturesthereof. In a preferred embodiment α-tocopherol is employed. Also calledvitamin E, α-tocopherol is not a typical lipid oil. It has a higherpolarity than most lipid oils, particularly triglycerides, and is notsaponifiable. It has practically no solubility in water.

[0085] In the second aspect, the invention is an tocopherol emulsion inthe form of a self-emulsifying system where the system is to be used forthe oral administration of water insoluble (or poorly water soluble orwater soluble agents modified to be less water soluble or mixturesthereof) drugs where that is desired. In this embodiment, an oil phasewith surfactant and drug or drug mixture is encapsulated into a soft orhard gelatin capsule. Suitable solidification agents with melting pointsin the range of 40 to 60° C. such as high molecular weight polyethyleneglycols (MW>1000) and glycerides such as those available under the tradename Gelucire (Gattefose Corp. Saint Priest, France) can be added toallow filling of the formulation into a hard gelatin capsule at hightemperature. Semi-solid formulations are formed upon room temperatureequilibration. Upon dissolution of the gelatin in the stomach andduodenum, the oil is released and forms a fine emulsion with a meandroplet diameter of between 2-5 microns spontaneously. The emulsion isthen taken up by the microvilli of the intestine and released into thebloodstream.

[0086] In a third aspect, the invention comprises microemulsionscontaining tocopherol preferably α-tocopherol Microemulsions refer to asub-class of emulsions where the emulsion suspension is essentiallyclear and indefinitely stable by virtue of the extremely small size ofthe oil/drug microaggregates dispersed therein.

[0087] In a fourth aspect of the invention, PEGylated vitamin E (TPGS)is used as a primary surfactant in emulsions of vitamin E. PEGylatedvitamin E is utilized as a primary surfactant, a stabilizer and also asa supplementary solvent in emulsions of vitamin E. Polyethylene glycol(PEG) is also useful as a co-solvent in the emulsions of this invention.Of particular use is polyethylene glycol 200, 300, 400 or mixturesthereof.

[0088] The α-tocopherol concentration of the emulsions of this inventioncan be from about 1 to about 10% w/v. The ratio of α-tocopherol to TPGSis optimally from about 1:1 to about 10:1 (w/w).

[0089] The emulsions of the invention may further include surfactantssuch as ascorbyl-6 palmitate; stearylamine; pegylated phospholipids,sucrose fatty acid esters and various vitamin E derivatives comprisingα-tocopherol nicotinate, tocopherol phosphate, and nonionic, syntheticsurfactant mixtures, such as polyoxypropylene-polyoxyethylene glycolnonionic block copolymer.

[0090] The emulsions of the invention can comprise an aqueous medium.The aqueous phase generally has an osmolality of approximately 300 mOsmand may include sodium chloride, sorbitol, mannitol, polyethyleneglycol, propylene glycol albumin, polypep and mixtures thereof. Thismedium can also contain various additives to assist in stabilizing theemulsion or in rendering the formulation biocompatible. Acceptableadditives include acidifying agents, alkalizing agents, antimicrobialpreservatives, antioxidants, buffering agents, chelating agents,suspending and/or viscosity-increasing agents, and tonicity agents.Preferably, agents to control the pH, tonicity, and increase viscosityare included. Optimally, a tonicity of at least 250 mOsm is achievedwith an agent which also increases viscosity, such as sorbitol orsucrose.

[0091] The emulsions of the invention for intravenous injection have aparticle size (mean droplet diameter) of 10 to 500 nm, preferably 10 to200 nm and most preferably 10 to 100 nm. For intravenous emulsions, thespleen and liver will eliminate particles greater than 500 mn in sizethrough the RES.

[0092] A preferred form of the invention includes paclitaxel, a verywater-insoluble cytotoxin used in the treatment of uterine cancer andother carcinomas. An emulsion composition of the present inventioncomprises a solution of vitamin E containing paclitaxel at aconcentration of up to 20 mg/mL, four times that currently available byprescription, and a biocompatible surfactant such that the emulsionmicrodroplets are less than 0.2 microns and are terminally sterilizableby filtration.

[0093] Preferred injectable compositions contain: 0.1-1.0% paclitaxel(1-10 mg/ml); 1-10% PEG400; 3-10% Vitamin E; 1-6% TPGS and 0.5-2.5%Pluronic F127.

[0094] Another preferred composition contains: 1.0 paclitaxel (10mg/ml), 6% PEG400, 8% Vitamin E, 5% TPGS, 1% Pluronic F127 and 80%aqueous solution.

[0095] Preferred formulations for self-emulsifying systems are asfollows: 0.1-20% paclitaxel, 10-90% Vitamin E, 10-90% PEG 400 orN-methyl-2-pyrrolidone, 5-50% TPGS, 5-50% a secondary hydrophilificsurfactant, such as Polysorbates (Tween 80), Pluronics (Pluronic F127)or Cremophor EL/RH40, Solutol HS-15). The oil phase (vitamin E) canoptionally contain polyvinylpyrrolidone, glycerol and propylene glycolesters such as mono-/di-/triglycerides and mono-diesters of propyleneglycol. In addition, high MW PEGs (1,000-10,000) and high melting pointglycerol esters can be included to provide the formulation withsemisolid consistency.

[0096] A further embodiment of the invention is a method of treatingcarcinomas comprising the parenteral administration of a bolus dose ofpaclitaxel in vitamin E emulsion with and without PEGylated vitamin E byintravenous injection once daily or every second day over a therapeuticcourse of several weeks. Such method can be used for the treatment ofcarcinomas of the breast, lung, skin and uterus.

[0097] The general principles of the present invention may be more fullyappreciated by reference to the following non-limiting examples.

EXAMPLES Example 1 Dissolution of Paclitaxel in α-tocopherol

[0098] α-Tocopherol was obtained from Sigma Chemical Company (St Louis,Mo.) in the form of a synthetic dl-α-tocopherol of 95% purity preparedfrom phytol. The oil was amber in color and very viscous. Paclitaxel waspurchased from Hauser Chemical Research (Boulder, Colo.), and was 99.9%purity by HPLC. Paclitaxel 200 mg was dissolved in 6 mL of dry absoluteethanol (Spectrum Chemical Manufacturing Corp, Gardenia, Calif.) andadded to 1 gm α-tocopherol. The ethanol was then removed by vacuum at42° C. until the residue was brought to constant weight. Independentstudies showed that the ethanol content was less than 0.3% (w/v).

[0099] The resultant solution was clear, amber and very viscous, with anominal concentration of 200 mg/gm (w/w) paclitaxel in α-tocopherol.Higher concentrations of paclitaxel (up to 400 mg/gm, w/w) can besolubilized in α-tocopherol.

Example 2 Anionic Surfactant Used to Prepare α-tocopherol Emulsions

[0100] Paclitaxel 2 gm in 10 gm of α-tocopherol, prepared as describedin Example 1, was emulsified with ascorbyl palmitate as thetriethanolamine salt by the following method. A solution consisting ofascorbic acid 20 mM was buffered to pH 6.8 with triethanolamine as thefree base to form 2× buffer. 50 mL of the 2× buffer was placed in aWaring blender. 0.5 gm of ascorbyl-6-palmitate (Roche Vitamins and FineChemicals, Nutley, N.J.), an anionic surfactant, was added and thesolution blended at high speed for 2 min at 40° C. under argon. Theα-tocopherol containing paclitaxel was then added into the blender withthe surfactant and buffer. Mixing was continued under argon until acoarse, milky, pre-emulsion was obtained, approximately after 1 min at40° C. Water for injection was then added, bringing the final volume to100 mL.

[0101] The pre-emulsion was transferred to the feed vessel of aMicrofluidizer Model 110Y (Microfluidics Inc, Newton, Mass.). The unitwas immersed in a bath to maintain a process temperature ofapproximately 60° C. during homogenization, and was flushed with argonbefore use. After priming, the emulsion was passed through thehomogenizer in continuous re-cycle for 10 minutes at a pressure gradientof about 18 kpsi across the interaction head. The flow rate was about300 mL/min, indicating that about 25 passes through the homogenizerresulted.

[0102] The resultant paclitaxel emulsion in an α-tocopherol vehicle wasbottled in amber vials under argon and stored with refrigeration at 7°C. and 25° C. Samples were taken at discrete time intervals for particlesizing and chemical analysis.

[0103] Data taken with a Nicomp Model 370 Submicron Particle Sizer(Particle Sizing Systems Inc, Santa Barbara, Calif.) showed that theemulsion had a mean particle diameter of 280 nm.

Example 3 Use of PEGylated Vitamin E (TPGS)

[0104] A ternary phase diagram was constructed for α-tocopherol,PEGylated vitamin E (TPGS, vitamin-Epolyoxyethyleneglycol-1000-succinate, obtained from Eastman ChemicalCo., Kingsport, Tenn.), and water. TPGS was first melted at 42° C. andmixed gravimetrically with α-tocopherol at various proportions from 1 to100% TPGS, the balance being α-tocopherol. Mixtures were miscible at allconcentrations. Water was then added to each mixture in such a way thatthe final water concentration was increased stepwise from zero to 97.5%.At each step, observations were made of the phase behavior of themixture. As appropriate, mixing was performed by vortexing andsonication, and the mixture was heated or centrifuged to assess itsphase composition.

[0105] A broad area of biphasic o/w emulsions suitable for parenteraladministration was found at water concentrations above 80%. Theemulsions formed were milky white, free flowing liquids that containeddisperse α-tocopherol microparticles stabilized by non-ionic surfactant.Also in this area, microemulsions potentially suitable as drug carrierswere observed at TPGS to oil ratios above about 1:1. At lower watercontent, a broad area containing transparent gels (reverse emulsions)was noted. Separating the two areas (high and low water content) is anarea composed of opaque, soap-like liquid crystals.

[0106] Phase diagrams of α-tocopherol with surfactant combinations, forexample TPGS with a nonionic, anionic or cationic co-surfactant (forexample glutamyl stearate, ascorbyl palmitate or Pluronic F-68), or drugcan be prepared in a similar manner.

Example 4 α-Tocopherol Emulsion for Intravenous Delivery of Paclitaxel

[0107] A formulation of the following composition was prepared:paclitaxel 1.0 gm % α-tocopherol 3.0 gm % TPGS 2.0 gm %Ascorbyl-6-Palmitate 0.25 gm % Sorbitol 5.0 gm % Triethanolamine to pH6.8 Water qs to 100 mL

[0108] The method of preparation was as follows: synthetic α-tocopherol(Roche Vitamins, Nutley, N.J.), paclitaxel (Hauser, Boulder, Colo.),ascorbyl 6-palmitate (Aldrich Chemical Co, Milwaukee, Wis.) and TPGSwere dissolved in 10 volumes of anhydrous undenatured, ethanol (SpectrumQuality Products, Gardenia, Calif.) with heating to 40-45° C. Theethanol was then drawn off with vacuum until no more than 0.3% remainedby weight.

[0109] Pre-warmed aqueous solution containing a biocompatible osmolyteand buffer were added with gentle mixing and a white milk formedimmediately. This mixture was further improved by gentle rotation for 10minutes with continuous warming at 40-45° C. This pre-mixture at aboutpH 7 was then further emulsified as described below.

[0110] The pre-mixture at 40-45° C. was homogenized in an Avestin C5homogenizer (Avestin, Ottawa Canada) at 26 Kpsi for 12 minutes at 44° C.The resultant mixture contained microparticles of α-tocopherol with amean size of about 200 nm. Further pH adjustment was made with analkaline 1 M solution of triethanolamine (Spectrum Quality Products).

[0111] In order to avoid gelation of the TPGS during the early stage ofemulsification, all operations were performed above 40° C. and care wastaken to avoid exposure of the solutions to cold air by covering allvessels containing the mixture. Secondly, less than 2% TPGS shouldgenerally be dissolved in α-tocopherol oil before pre-emulsification,the balance of the TPGS being first dissolved in the aqueous bufferbefore the pre-emulsion is prepared. The solution gels at concentrationsof TPGS higher than 2%.

[0112] Physical stability of the emulsion was then examined by placingmultiple vials on storage at 4° C. and 25° C. Over several months, vialswere periodically withdrawn for particle sizing. Mean particle size, asdetermined with the Nicomp Model 370 (Particle Sizing Systems, SantaBarbara, Calif.), is shown for the two storage temperatures in FIG. 1.The particle size distribution was bi-modal.

Example 5 Chemical Stability of Paclitaxel in an α-tocopherol Emulsion

[0113] After emulsification, the formulation of Example 4 was analyzedfor paclitaxel on a Phenosphere CN column (5 microns, 150×4.6 mm). Themobile phase consisted of a methanol/water gradient, with a flow rate of1.0 mL/min. A UV detector set at 230 nm was used to detect andquantitate paclitaxel. A single peak was detected (FIG. 2), which had aretention time and mass spectrogram consistent with native referencepaclitaxel obtained from Hauser Chemical (Boulder, Colo.).

[0114] Chemical stability of the emulsion of example 4 was examined byHPLC during storage. The data of FIG. 3 demonstrate that paclitaxelremains stable in the emulsion for periods of at least 3 months,independent of the storage temperature. Taken together, the data ofFIGS. 2 and 3 demonstrate successful retention of drug potency andemulsion stability when stored at 4° C. for a period of at least 3months.

Example 6 Paclitaxel Emulsion Formulation QWA

[0115] An emulsion of paclitaxel 10 mg/ml for intravenous drug delivery,having the following composition, was prepared as described in Example4. paclitaxel 1.0 gm % α-tocopherol 3.0 gm % TPGS 1.5 gm %Ascorbyl-6-Palmitate 0.25 gm % Sorbitol 4.0 gm % Triethanolamine to pH6.8 Water qs to 100 mL

Example 7 Paclitaxel Emulsion Formulation QWB

[0116] A second emulsion of paclitaxel 10 mg/ml for intravenous drugdelivery, having the following composition, was prepared as described inExample 4. paclitaxel 1.0 gm % α-tocopherol 3.0 gm % TPGS 1.5 gm %Solutol HS-15 1.0 gm % Sorbitol 4.0 gm % Triethanolamine to pH 6.8 Waterqs to 100 mL

Example 8 10 mg/mL Paclitaxel Emulsion Formulation QWC

[0117] A third emulsion formulation of paclitaxel 10 mg/ml was preparedas follows using Poloxamer 407 (BASF Corp, Parsippany, N.J.) as aco-surfactant. paclitaxel 1.0 gm % α-tocopherol 6.0 gm % TPGS 3.0 gm %Poloxamer 407 1.0 gm % Sorbitol 4.0 gm % Triethanolamine to pH 6.8 Waterfor injection qs to 100 mL

[0118] In this example, 1.0 gm Poloxamer 407 and 1.0 gm paclitaxel weredissolved in 6.0 gm α-tocopherol with ethanol 10 volumes and gentleheating. The ethanol was then removed under vacuum. Separately, anaqueous buffer was prepared by dissolving 3.0 gm TPGS and 4.0 gmsorbitol in a final volume of 90 mL water for injection. Both oil andwater solutions were warmed to 45° C. and mixed with sonication to makea pre-emulsion. A vacuum was used to remove excess air from thepre-emulsion before homogenization.

[0119] Homogenization was performed in an Avestin C5 as alreadydescribed. The pressure differential across the homogenization valve was25 kpsi and the temperature of the feed was 42°-45° C. A chiller wasused to ensure that the product exiting the homogenizer did not exceed atemperature of 50° C. Flow rates of 50 mL/min were obtained duringhomogenization. After about 20 passes in a recycling mode, the emulsionbecame more translucent. Homogenization was continued for 20 min.Samples were collected and sealed in vials as described before. A fineα-tocopherol emulsion for intravenous delivery of paclitaxel wasobtained. The mean particle diameter of the emulsion was 77 nm.Following 0.22μ sterile filtration through a 0.22 micron Durapore filter(Millipore Corp, Bedford, Mass.), the emulsion was filled in vials andstored at 4° C. until used for intravenous injection.

Example 9 5 mg/mL Paclitaxel Emulsion Formulation QWC

[0120] An additional emulsion of paclitaxel was prepared as described inExample 8 but incorporating 5 instead of 10 mg/ml of the drug. Thecomposition of this emulsion is as follows: paclitaxel 0.5 gm %α-tocopherol 6.0 gm % TPGS 3.0 gm % Poloxamer 407 1.0 gm % Sorbitol 4.0gm % Triethanolamine to pH 6.8 Water for injection qs to 100 mL

[0121] Following homogenization as described in example 8, a somewhattranslucent emulsion of α-tocopherol and paclitaxel with a mean particlediameter of 52 nm was obtained. Following sterile filtration through a0.22 micron Durapore filter (Millipore Corp, Bedford, Mass.), theemulsion was filled in vials and stored at 4° C. until used forintravenous injection. Drug losses on filtration were less-than 1%.

Example 10 Paclitaxel Emulsion Formulation QWD

[0122] A fifth emulsion of α-tocopherol for intravenous administrationof paclitaxel was prepared as follows: paclitaxel 0.5 gm % α-tocopherol6.0 gm % TPGS 3.0 gm % Poloxamer 407 1.5 gm % Polyethyleneglycol 200 0.7gm % Sorbitol 4.0 gm % Triethanolamine to pH 6.8 Water for injection qsto 100 mL

[0123] Synthetic α-tocopherol USP-FCC obtained from Roche Vitamins(Nutley, N.J.) was used in this formation. Polyethyleneglycol 200(PEG-200) was obtained from Sigma Chemical Co.

[0124] Following homogenization, a somewhat translucent emulsion with amean particle diameter of 60 nm was obtained. Following 0.22μ sterilefiltration through a 0.22 micron Durapore filter (Millipore Corp,Bedford, Mass.), the emulsion was filled in vials and stored at 4° C.until used for intravenous injection. Drug losses on filtration wereless than 1%.

Example 11 Dissolution of Paclitaxel in TPGS and Preparation of MicellarSolutions

[0125] We observed good solubility of paclitaxel in TPGS, about 100 mgdrug per 1.0 gm of TPGS. Micellar solutions of TPGS containingpaclitaxel were prepared as follows. A stock solution of paclitaxel inTPGS was made up by dissolving 90 mg paclitaxel in 1.0 gm TPGS at 45° C.with ethanol, which was then removed under vacuum. Serial dilutions werethen prepared by diluting the paclitaxel stock with additional TPGS toobtain paclitaxel in TPGS at concentrations of 0.1, 1, 5, 10, 25, 50, 75and 90 mg/mL. Using fresh test tubes, 100 mg of each paclitaxelconcentration in TPGS was dissolved in 0.9 mL water. All test tubes weremixed by vortex and by sonication at 45° C. Clear micellar solutions inwater were obtained corresponding to final paclitaxel concentrations of0.01, 0.1, 0.5, 1.0, 2.5, 5.0, 7.5 and 9.0 mg/mL.

[0126] A Nicomp Model 370 laser particle sizer (Particle Sizing Systems,Santa Barbara, Calif.) was used to examine the solutions. Particle sizeson the order of 10 nm were obtained, consistent with the presence ofmicelles of TPGS and paclitaxel.

[0127] Micellar solutions of paclitaxel in TPGS containing up to 2.5mg/mL paclitaxel were stable for at least 24 hr whereas those at 5.0,7.5 and 9.0 mg/mL were unstable and drug crystals formed rapidly andirreversibly. These observations imply that paclitaxel remainssolubilized only in the presence of an α-tocopherol-rich domain withinthe emulsion particles. Thus, an optimum ratio of α-tocopherol to TPGSis needed in order to produce emulsions in which higher concentrationsof paclitaxel can be stabilized.

[0128] When adjusted to the proper tonicity and pH, micellar solutionshave utility for slow IV drip administration of paclitaxel to cancerpatients, although the AUC is expected to be low.

[0129] The utility of TPGS in α-tocopherol emulsions is a synergy ofseveral desirable characteristics. First, it has its own affinity forpaclitaxel, probably by virtue of the α-tocopherol that makes up thehydrophobic portion of its molecular structure. Secondly, interfacialtension of TPGS in water with α-tocopherol is about 10 dynes/cm,sufficient to emulsify free α-tocopherol, especially when used with aco-surfactant. Third, polyoxyethylated surfactants such as TPGS, havewell established, superior properties as a “stealth coat” for injectableparticles, by dramatically reducing trapping of the particles in theliver and spleen, as is well known in the art. But the unexpected andunique finding with TPGS as a surfactant for α-tocopherol emulsions, wasthe finding of all three desirable characteristics in a single molecule.An additional advantage of TPGS is the fact that it forms stableself-emulsifying systems in mixtures with oils and solvents such aspropylene glycol and polyethylene glycol, suggesting a synergy when usedwith α-tocopherol for oral drug delivery.

[0130] When adjusted to the proper tonicity and pH, micellar solutionshave utility for slow IV drip administration of paclitaxel to cancerpatients, although the AUC is expected to be low.

Example 12 20 mg/mL Paclitaxel Emulsion Formulation

[0131] A coarse, emulsion containing 20 mg/mL paclitaxel in α-tocopherolwas obtained with 5% α-tocopherol and 5% TPGS by the methods describedin Example 4, simply by increasing the concentrations. No effort wasmade to test higher concentrations simply because no further increase isnecessary for clinically useful intravenous emulsions.

Example 13 Use of Other PEG Surfactants in α-tocopherol Emulsions

[0132] A variety of other pegylated surfactants, for example TritonX-100, PEG 25 propylene glycol stearate, Brij 35 (Sigma Chemical Co),Myrj 45, 52 and 100, Tween 80 (Spectrum Quality Products), PEG 25glyceryl trioleate (Goldschmidt Chemical Corp, Hopewell, Va.), haveutility in emulsifying α-tocopherol.

[0133] However, experiments with some other pegylated surfactants failedto convincingly stabilize paclitaxel in an α-tocopherol emulsion. Todemonstrate the unique utility of TPGS, three emulsions were prepared asdescribed in Example 9, but Tween 80 and Myrj 52 were substituted forTPGS as the primary surfactant in separate emulsions. These twosurfactants were chosen because Tween 80 and Myrj 52 have HLB valuesessentially equivalent to TPGS and make reasonably good emulsions ofα-tocopherol. However, when 5 mg/mL paclitaxel was included in theformulation, drug crystallization was noted very rapidly afterpreparation of the pre-emulsion, and the processed emulsions of Tween 80and Myrj 52 were characterized as coarse, containing rod-shapedparticles up to several microns in length, consistent with crystals ofpaclitaxel. Unlike the TPGS emulsion, which passed readily through a0.22 micron filter with less than 1% loss of drug, the Tween and Myrjemulsions were unfilterable because of the presence of this crystallinedrug material.

[0134] There are several possible explanations for the unexpectedimprovement of the α-tocopherol paclitaxel emulsions with TPGS. The drughas good solubility in TPGS, up to about 100 mg/mL. Most likely it isthe strength of the affinity of paclitaxel benzyl side chains with theplanar structure of the α-tocopherol phenolic ring in the TPGS moleculethat stabilizes the complex of drug and carrier. In addition thesuccinate linker between the α-tocopherol and PEG tail is a novelfeature of this molecule that distinguishes its structure from otherPEGylated surfactants tested.

Example 14 Poloxamer-based α-tocopherol Emulsion

[0135] α-tocopherol 6.0 gm % Poloxamer 407 2.5 gm % Ascorbyl Palmitate0.3 gm % Sorbitol 6.0 gm % Triethanolamine to pH 7.4 Water qs to 100 mL

[0136] An α-tocopherol emulsion was prepared using Poloxamer 407 (BASF)as the primary surfactant. The white milky pre-mixture was homogenizedwith continuous recycling for 10 minutes at 25 Kpsi in a C5 homogenizer(Avestin, Ottawa Canada) with a feed temperature of 45° C. and a chillerloop for the product out set at 15° C. A fine, sterile filterableemulsion of α-tocopherol microparticles resulted. However, when thisformulation was made with paclitaxel, precipitation of the paclitaxelwas noted following overnight storage in the refrigerator, againunderlying the superior utility of TPGS as the principle surfactant.

Example 15 Lyophilized Emulsion Formulation

[0137] Maltrin M100 (Grain Processing Corporation, Muscatine, Iowa) wasadded as a 2× stock in water to the emulsion of Example 14. Aliquotswere then frozen in a shell freezer and lyophilized under vacuum. Onreconstitution with water, a fine emulsion was recovered.

[0138] Lyophilized formulations have utility where the indefinite shelflife of a lyophilized preparation is preferred. Lyophilizableformulations containing other saccharides, such as mannitol, albumin orPolyPep from Sigma Chemicals, St. Louis, Mo. can also be prepared.

Example 16 In vitro Release of Paclitaxel from α-tocopherol Emulsions

[0139] One of the desired characteristics of a drug delivery vehicle isto provide sustained release of the incorporated drug, a characteristicquite often correlated with improved pharmacokinetics and efficacy. Inparticular, long-circulating emulsions of paclitaxel can improve thedelivery of the drug to cancer sites in the body. We have surprisinglyfound that the emulsions of the present invention do provide sustainedrelease of paclitaxel when compared to the only FDA-approved formulationof paclitaxel at this time [Taxol®, Bristol Myers Squibb (BMS),Princeton, N.J.]. Emulsions were prepared having paclitaxelconcentrations of 6 mg/mL (QWA) and 7 mg/mL (QWB). For comparison, Taxolcontains 6 mg/ml of paclitaxel dissolved in ethanol:cremophore EL 1:1(v/v). In vitro release of paclitaxel from the different formulationsinto a solution of phosphate-buffered saline (PBS) at 37° C. wasmonitored using a dialysis membrane that is freely permeable topaclitaxel (MW cut-off of 10 kilodaltons). Quantification of the drug inpre- and post-dialysis samples was performed by HPLC. Drug releaseprofiles in terms of both percent release and concentration ofpaclitaxel released over time were generated. As can be seen from thedata in FIG. 4, less than 5% of paclitaxel was dialyzed from theemulsions over 24 hr, whereas about 12% was recovered outside thedialysis bag from the marketed BMS formulation. This indicates that drugrelease from the emulsion was significantly slowed relative to thecommercially available solution.

Example 17 Biocompatibility of α-tocopherol Emulsions ContainingPaclitaxel

[0140] An acute single-dose toxicity study was performed. Mice 20-25 gmeach were purchased and acclimatized in an approved animal facility.Groups of mice (n=3) received doses of the formulation containing from30 to 90 mg/kg paclitaxel in the α-tocopherol emulsion prepared asdescribed in Example 6. All injections were given intravenously bytailvein bolus.

[0141] Although all injections were given by bolus IV push, no deaths orimmediate toxicity were observed at any dose, even at 90 mg/kg. Theresults for body weight are shown in Table 2. Weight loss was 17% in thehighest group but all groups, even at 90 mg/kg, recovered or gained bodyweight over a period of 10 days post injection.

[0142] A vehicle toxicity study was also done. Animals receivingdrug-free emulsion grew rapidly, and gained slightly more weight thananimals receiving saline or not injected. This was attributed to thevitamin and calorie content of the formulation.

[0143] We observed a maximal tolerable dose (MTD) for paclitaxel ofgreater than 90 mg/kg (Table 2), with no adverse reactions noted. Thisis more than double the best literature values reported, in which deathswere observed at much smaller doses. The FDA-approved formulation ofTaxol® causes death in mice at bolus intravenous doses of 10 mg/kg, afinding repeated in our hands. In the rat, Taxol® was uniformly fatal atall dilutions and dose regimes we tested. In contrast, the compositionof Example 6 was well tolerated in rats, and is even improved overTaxotere, a less toxic paclitaxel analogue commercially marketed byRhone-Poulenc Rorer.

[0144] One possible explanation for the high drug tolerance is that theemulsion is behaving as a slow-release depot for the drug as suggestedfrom the in vitro release data in Example 16. TABLE 2 Average BodyWeight Change of Mice Treated with Paclitaxel Emulsion Treatment Numberof BW Change (gm) (dose, mg/kg Animals Day 2 Day 7 Saline 4 1.0 3.4Vehicle 4 1.2 3.5 Paclitaxel Emulsion 2 −1.0 2.2 (QWA) (36.3) PaclitaxelEmulsion 4 −1.8 1.7 (QWA) (54.4) Paclitaxel Emulsion 4 −1.5 1.6 (QWA)(72.6) Paclitaxel Emulsion 1 −1.6 (QWA) (90.7)

Example 18 Efficacy Evaluation of Paclitaxel Emulsion

[0145] The paclitaxel emulsion of Example 6 was also evaluated forefficacy against staged B16 melanoma tumors in nude mice and the data isshown in Table 3. Once again, the marketed product Taxol® was used as areference formulation. Tumor cells were administered subcutaneously andtherapy started by a tail vein injection at day 4 post-tumoradministration at the indicated dosing schedule. Efficacy was expressedas percent increase in life-span (% ILS).

[0146] The following conclusions can be drawn from the data in Table 3:a) an increased life span of about 10% was obtained by administration ofBMS Taxol at 10 mg/kg Q2Dx4, b) % ILS values improved to 30-50% byadministration of the α-tocopherol emulsion of paclitaxel at 30, 40 or50 mg/kg Q2Dx4, dose levels made possible by the higher MTD, c) a nicedose response was observed when the emulsion was administered at 30, 50and 70 mg/kg Q4Dx3, with about 80% ILS being observed at 70 mg/kg and,d) even at 90 mg/kg dosed only once at day 4, there was about 36% ILS.These data clearly illustrate the potential of the emulsions of thepresent invention to substantially improve the efficacy of paclitaxel.

Example 19 Efficacy Evaluation of Paclitaxel Emulsions

[0147] The emulsions of examples 6, 7 and 8 (QWA, QWB and QWCrespectively) were compared for efficacy against B16 melanoma in mice;Taxol® was again used as a reference formulation. Methods essentiallyidentical to those of Example 18 were used. The data from this study issummarized in Table 3. Efficacy was expressed as: a) percent tumorgrowth inhibition (% T/C, where T and C stand for treated and controlanimals, respectively); b) tumor growth delay value (T-C), and c) logcell kill which is defined as the ratio of the T-C value over 3.32×tumordoubling time. The latter parameter for this particular tumor model wascalculated to be 1.75 days. As can be seen from the results in Table 4,all measures of efficacy: tumor growth inhibition, tumor growth delayvalue and log cell kill demonstrate superior efficacy of α-tocopherolemulsions as a drug delivery vehicle over Taxol®, particularly when theemulsions were dosed every four days at 70 mg/kg. As explained inExample 16, this increased efficacy is likely a result of improved drugbiocompatibility and/or sustained release. TABLE 3 Survival of Mice withB16 Tumors Treated with QWA and Taxol ® Mean Survival Time, Days %ILS^(b) (vs vehicle) Treatment Group & Schedule (Mean ± S.E.M^(a)) (Mean± S.E.M) Vehicle Control (Days 4, 13.2 ± 0.9 — 8, 12) Saline Control(Days 4, 8, 15.8 ± 1.2 19.7 ± 8.6 12) Taxol ® (10 mg/kg) (Days 4, 16.4 ±0.7 24.2 ± 5.4 6, 8, 10) QWA (30 mg/kg) (Days 4, 6, 19.2 ± 1.4  45.4 ±10.3 8) QWA (40 mg/kg) (Days 4, 6, 21.3 ± 1.4  61.4 ± 10.3 8) QWA (50mg/kg) (Days 4, 6, 18.8 ± 0.7 42.4 ± 5.7 8) QWA (30 mg/kg) (Days 4, 8,15.3 ± 0.8 15.9 ± 6.4 12) QWA (50 mg/kg) (Days 4, 8, 20.7 ± 1.3 56.8 ±9.5 12) QWA (70 mg/kg) (Days 4, 8, 24.2 ± 0.9 83.3 ± 6.4 12) QWA (90mg/kg) (Day 4 only) 18.0 ± 0.6 36.4 ± 4.4

[0148] according to the NCI standards an ILS value greater than 50%indicates significant anti-tumor activity. TABLE 4 Comparison of 3paclitaxel emulsions and Taxol against early-stage B16 melanoma Mediantumor Median % Log Dosing Total wt. on day tumor wt. T/C cell TestDosage Schedule Dose 15 on day 18 Day T − C kill Article mg/kg/day(days) (mg/kg) (mg) mg (range) 15 (days) total Control 0 4, 6, 8, 10 0836 2139 — — — Taxol ® 20 4, 6, 8, 10 80 383 1217 46 2 0.34 QWA 20 4, 6,8, 10 80 381 1197 46 2 0.34 QWA 40 4, 6, 8, 10 160 104 306 12 7 1.2 QWA70 4, 8, 12, 16, 350 15 11 ˜2 20 QWB 20 80 197 653 24 5 0.86 QWB 30 4,6, 8, 10 120 139 449 17 5 0.86 QWB 40 4, 6, 8, 10 Toxic QWC 20 80 319848 34 3 0.52 QWC 40 4, 6, 8, 10 160 53 194 6 8 1.4 QWC 70 4, 6, 8, 10350 33 66 4 >15 >2.6 4, 8, 12, 16, 20

Example 20 Self-emulsification of an α-tocopherol/Tagat TO Mixture

[0149] α-tocopherol 2.0 gm and Tagat TO (Goldschmidt Chemical Corp,Hopewell, Va.) 800 mg were dissolved together. About 80 mg of the oilymixture was transferred to a test tube and water was then added. Withgentle hand mixing, there was immediate development of a rich milkyemulsion, consistent with “self-emulsifying systems” proposed as drugdelivery systems, in which surfactant-oil mixtures spontaneously form anemulsion upon exposure to aqueous media.

Example 21 Self-emulsifying Formulation Containing Paclitaxel

[0150] Paclitaxel 50 mg/ml was prepared in α-tocopherol by the methoddescribed in Example 1. Tagat TO 20% (w/w) was added. The resultantmixture was clear, viscous and amber in color. A 100 mg quantity of theoily mixture was transferred to a test tube. On addition of 1 mL ofwater, with vortex mixing, a fine emulsion resulted.

Example 22 Self-emulsifying Formulation of Paclitaxel

[0151] Paclitaxel 50 mg/ml was prepared in α-tocopherol by the methoddescribed in Example 1. After removal of the ethanol under vacuum, 20%TPGS and 10% polyoxyethyleneglycol 200 (Sigma Chemical Co) were added byweight. A demonstration of the self-emulsification ability of thissystem was then performed by adding 20 mL of deionized water to 100 mgof the oily mixture at 37° C. Upon gentle mixing, a white, thin emulsionformed, consisting of fine emulsion particles demonstrated with theMalvern Mastersizer (Malvern Instruments, Worcester, Mass.) to have amean size of 2 microns, and a cumulative distribution 90% of which wasless than 10 microns.

Example 23 Etoposide Emulsion Formulation in α-tocopherol

[0152] Etoposide 4 mg (Sigma Chemical Co) was dissolved in the followingsurfactant-oil mixture: Etoposide  4 mg α-tocopherol 300 mg  TPGS 50 mgPoloxamer 407 50 mg

[0153] Ethanol and gentle warming was used to form a clear ambersolution of drug in oil. The ethanol was then removed under vacuum.

[0154] A pre-emulsion was formed by adding 4.5 mL of water containing 4%sorbitol and 100 mg TPGS at 45° C. with sonication. The particle sizewas further reduced by processing in an Emulsiflex 1000 (Avestin, OttawaCanada). The body of the Emulsiflex 1000 was fitted with a pair of 5 mLsyringes and the entire apparatus heated to 45° C. before use. The 5 mLof emulsion was then passed through it by hand approximately 10 times. Afree flowing, practical emulsion of etoposide in an α-tocopherol vehicleresulted.

[0155] We note that the solubilized form of etoposide in α-tocopherolcan also be used as an oral dosage form by adaption of the methods ofthe preceding examples.

Example 24 Dissolution of Ibuprofen or Griseofulvin in α-tocopherol

[0156] Ibuprofen is a pain-killer, and may be administered by injectionwhen required if there is danger that the drug will irritate thestomach. The following solution of ibuprofen in α-tocopherol may beemulsified for intravenous administration.

[0157] Ibuprofen (Sigma Chemicals), 12 mg. crystalline, dissolvedwithout solvent in α-tocopherol, 120 mg, by gentle heating. Theresultant 10% solution of Ibuprofen in vitamin E can be emulsified bythe method s described in Examples 4, 6, 7, 8 or 22.

[0158] An antifungal compound, griseofulvin, 12 mg, was first dissolvedin 3 mL of anhydrous ethanol; α-tocopherol was then added, 180 mg, andthe ethanol was removed with gentle heating under vacuum. The resultantsolution of griseofulvin in α-tocopherol is clear and can be emulsifiedby the methods described in Examples 4, 6, 7, 8 or 22.

Example 25 Vitamin E Succinate Emulsion Formulation

[0159] Vitamin E succinate has been suggested as a therapeutic for thetreatment of lymphomas and leukemias and for the chemoprevention ofcancer. The following is a composition and method for the emulsificationof vitamin E succinate in α-tocopherol. Sucrose ester S1170 is a productof Mitsubishi Kagaku Foods Corp, Tokyo Japan. Vitamin E succinate, asthe free acid, was obtained as a whitish powder from ICN Biomedicals,Aurora, Ohio. Emulsions incorporating other surfactants such aspluronics, and TPGS along with α-tocopherol and α-tocopherol succinatecan be prepared in a similar manner with and without a therapeuticagent.

[0160] α-Tocopherol 8 gm and vitamin E succinate 0.8 gm were dissolvedtogether in ethanol in a round bottom flask. After removal of thesolvent, 100 mL of an aqueous buffer was added. The alkaline bufferconsisting of 2% glycerol, 10 mM triethanolamine, and 0.5 gm % sucroseester S1170. After mixing for 2 min, the pre-emulsion was transferred toan Avestin Model C-5 homogenizer and homogenization was continued forabout 12 minutes at a process feed temperature of 58° C. The pressuredifferential across the interaction head was 25 to 26 kpsi. Duringhomogenization, pH was carefully monitored, and adjusted as required topH 7.0. Care was taken to exclude oxygen during the process. A finewhite emulsion resulted.

Example 26 α-tocopherol Levels in Esters

[0161] Levels of α-tocopherol in commercially available esters:tocopherol-acetate, -succinate, -nicotinate, -phosphate and TPGS wereeither provided by the vendor or determined by HPLC. The concentrationof free α-tocopherol in these solutions is less than 1.0%, generallyless than 0.5%.

Example 27 Resveratrol Emulsion Formulation

[0162] Resveratrol is a cancer chemopreventative first discovered as anextract of grape skins. It has been proposed as a dietary supplement.

[0163] Resveratrol was obtained from Sigma Chemical Co. While itdissolved poorly in ethanol, upon addition of 10 mg resveratrol, 100 mgof α-tocopherol, 100 mg TPGS and ethanol, a clear solution formedrapidly. Upon removal of the ethanol, a clear amber oil remained.

[0164] The oily solution of resveratrol can be formulated as aself-emulsifying system for oral delivery by the various methods of thepreceding examples.

Example 28 Muramyl Dipeptide Formulation

[0165] Muramyl dipeptides are derived from mycobacteria and are potentimmunostimulants representative of the class of muramyl peptides,mycolic acid and lipopolysaccharides. They have use, for example, in thetreatment of cancer, by stimulating the immune system to target andremove the cancer, particularly in connection with anti-cancer vaccines.More recently, muroctasin, a synthetic analog, has been proposed toreduce non-specific side effects of the bacterial wall extracts.

[0166] N-acetylmuramyl-6-O-steroyl-1-alanyl-d-isoglutamine was purchasedfrom Sigma Chemical Co. and 10 mg was dissolved in 100 mg α-tocopheroland 80 mg TPGS. Ethanol was used as a co-solvent to aid in dissolutionof the dipeptide, but was removed by evaporation under vacuum, leaving aclear solution in α-tocopherol and surfactant.

[0167] This oil solution of the drug can be emulsified for parenteraladministration by the various methods of the preceding examples.

Example 29 Alcohol-containing Emulsion

[0168] In attempting to adapt the teachings of PCT WO 95/11039 to theoral administration of paclitaxel, the following formulation was made.paclitaxel 0.125 gm α-tocopherol 0.325 gm TPGS 0.425 gm Ethanol 0.125 gm

[0169] As before, paclitaxel was dissolved in a α-tocopherol and TPGSwith ethanol, which was then removed under vacuum. By dry weight,residual ethanol was less than 3 mg (0.3% w/w). Fresh anhydrous ethanol0.125 gm was then added back to the formulation. After mixing, thesuitability of the formulation for oral administration, as in a gelatincapsule, was simulated by the following experiment. An aliquot of 100 mgof the free-flowing oil was added to 20 mL of water at 37° C. and mixedgently with a vortex mixer. A fine emulsion resulted. But after twentyminutes, microscopy revealed the growth of large numbers of crystals inrosettes, characteristic of paclitaxel precipitation. It was concludedthat this formulation was not suitable for oral administration ofpaclitaxel because large amounts of the drug would be in the form ofcrystals on entry into the duodenum, where it would be prevented fromuptake because of its physical form. We speculate that the excess ofethanol, in combination with the high ratio of TPGS to α-tocopherol, isresponsible for the observed crystallization of the drug from thisformulation.

Example 30 Alcohol-containing α-tocopherol Emulsion

[0170] In attempting to adapt the teachings of PCT WO 95/11039 to theintravenous administration of paclitaxel, the following formulation wasmade: paclitaxel 0.050 gm α-tocopherol 0.100 gm Lecithin 0.200 gmEthanol 0.100 gm Butanol 0.500 gm

[0171] As before, paclitaxel was dissolved in α-tocopherol and TPGS withethanol, which was then removed under vacuum. By dry weight, residualethanol was less than 2 mg (0.5% w/w). Fresh anhydrous ethanol 0.100 gmand n-butanol 0.500 gm was then added back to the formulation. A clearoil resulted. The injection concentrate was tested for biocompatibilityin administration by standard pharmaceutical practice of admixture withsaline. About 200 mg of the oil was placed into 20 mL of saline andmixed. Large flakes of insoluble material developed immediately and thegreatest amount of material formed dense deposits on the walls of thetest tube. The mixture was clearly unsuitable for parenteraladministration by any route, and we speculate that this is so regardlessof the identity of the drug contained in the formulation. By trial anderror we have learned that lecithin is a poor choice as surfactant forα-tocopherol by virtue of its low HLB (around 4). Other successfulexamples described here for fine emulsions suitable for parenteraladministration were all made with high HLB surfactants. Thesesurfactants include TPGS (HLB around 17), Poloxamer 407 (HLB about 22)and Tagat TO (HLB about 14.0). In general, we found that α-tocopherolemulsification is best performed with principal surfactants of HLB>10,preferably greater than 12. Lecithin is not in this class, although itcould be used as a co-surfactant. In comparison, typical o/w emulsionsof triglycerides are made with surfactants of HLB between 7 and 12,demonstrating that α-tocopherol emulsions are a unique class by virtueof the polarity and extreme hydrophobicity of the α-tocopherol, factorsthat also favor the solubility of lipophilic and slightly polarlipophilic drugs in α-tocopherol. See Emulsions: Theory and Practice,2nd Ed. p.248 (1985).

Example 31

[0172] Various formulations useful in the invention (Table 5) areprepared as follows: TABLE 5 B A (all surfactant in (split surfactant)oil) Composition of Injectable Weight Weight Weight Weight PaclitaxelEmulsions (g) (%) (g) (%) Oil Phase Paclitaxel 0.50 0.51 0.53 0.52 PEG400 6.02 6.04 6.38 6.30 TPGS 3.78 3.80 5.32 5.25 Pluronic 1.07 1.05 F127Vitamin E 8.04 8.07 8.51 8.40 Aqueous Phase TPGS 1.25 1.26 Pluronic 1.011.01 F127 Water 79.00 79.31 79.50 78.48 Total 99.60 100.00 101.30 100.00

[0173] Formulation A—Split Surfactants:

[0174] 1) 1.25 g TPGS and 1.01 g Pluronic F127 were dissolved in 79.00 gwater for injection by heating and stirring.

[0175] 2) 0.533 g paclitaxel was dissolved in 6.354 g PEG 400 by mixing(low shear) at 75° C.

[0176] 3) 3.992 g TPGS and 8.490 g Vitamin E were added and mixed (lowshear) at 45° C. until TPGS was melted and the mixture was visiblyhomogeneous. This oil phase represents a slight excess in order toaccount for incomplete transfer in Step 4.

[0177] 4) The aqueous phase (step 1) was heated to 45° C. and mixed atmedium shear (laboratory mixing motor) while 45° C. oil phase (step 2+3)was poured in over 1 minute. Mixing was continued 2 minutes more to forma crude emulsion.

[0178] 5) The emulsion was homogenized in an Avestin C5 in continuousrecycle mode for 1 hour at 22 Kpsi peak stroke pressure.

[0179] 6) Actual amounts and percentages shown in the table arecorrected for the incomplete transfer of oil phase during Step 4.

[0180] This method utilizing the split surfactants is useful in thecases where the solubility of a particular surfactant in the oil phaseis limited.

[0181] Formulation B—All Surfactants in Oil Phase

[0182] 1) 1.066 g paclitaxel was dissolved in 12.887 g PEG400 by mixing(low shear at 75° C.

[0183] 2) 10.739 g TPGS and 2.157 g Pluronic F127 were added and mixed(low shear) at 50-60° C. until both surfactants were completelymelted/dissolved.

[0184] 3) 17.176 g Vitamin E was added and mixed (low shear) at 45-50°C. until the mixture was visibly homogeneous.

[0185] 4) 21.8 g of the oil phase produced in Steps 1-4 was added over 1minute to 79.5 g water while mixing at medium shear (laboratory mixingmotor). Mixing was continued for a total of 3 minutes to form a crudeemulsion.

[0186] 5) Emulsion was homogenized in an Avestin C5 in continuousrecycle mode for 30 minutes at 22 K psi peak stroke pressures

[0187] From a processing perspective it is advantageous to have all ofthe surfactants in the oil phase. Both the dispersion of thepre-emulsion and subsequent homogenization are facilitated and potentialgellation of high melting point surfactants, such as TPGS, can beavoided.

Example 32 Etoposide Emulsion

[0188] A vitamin E emulsion (6.0% vitamin E, 3.5% TPGS, 6.0%, PEG400, 8%Pluronic F-127) and incorporating 2 mg/ml of Etoposide was prepared asfollows:

[0189] 1) 0.1044 g of Etoposide was dissolved in 3.1435 g of PEG 400 (5min at 65° C.).

[0190] 2) 2.0447 g of TPGS and 3.1563 g of Vitamin E were added andmixed until complete dissolution.

[0191] 3) The oil phase was mixed at 44° C. with 42.4 g of water forinjection incorporating 0.5 g of Pluronic F-127 (the aqueous phase wasdegassed by boiling prior to its mixing with the oil phase) and thepre-emulsion was formed by brief sonication.

[0192] 4) Upon homogenization in an Avestin CS at 22-24 Kpsi a fineemulsion was formed.

Example 33 Etoposide Emulsion

[0193] An α-Tocopherol emulsion containing PEG 300 and incorporating 2mg/ml of Etoposide was prepared as follows:

[0194] Etoposide was first dissolved in PEG-300 (10 min at 72° C.). TPGSand Vitamin E were then added to the drug solution. Aqueous phase (WFIcontaining Poloxamer 407) was degassed by boiling prior to use.Pre-emulsion was prepared by adding 5 g of the oil phase to 45 g ofwater at 45° C. After a 3-min mixing the pre-emulsion was homogenized at25 Kpsi for 30 min to produce a fine emulsion. The final composition ofthe emulsion is shown below: Component Composition (%, w/w) Etoposide0.2 Vitamin E 3.0 TPGS 1.5 PEG-300 3.0 Poloxamer 407 1.0 WFI (water forinjection) 92.3

Example 34

[0195] Additional paclitaxel emulsions for injection are presented inTable 6. TABLE 6 Composition of Injectable paclitaxel emulsions D E C(all surfactant in (all surfactant in Composition of (split surfactant)oil) oil) Injectable Paclitaxel Weight Weight Weight Weight WeightWeight Emulsions (g) (%) (g) (%) (g) (%) Oil Phase Paclitaxel 2.0 0.40.55 1.1 0.5 0.5 PEG 400 32.0 6.4 3.36 6.7 10.0 10.0 TPGS 18.85 3.772.60 5.2 4.3 4.3 Pluronic 0.52 1.0 5.1 1.1 F127 Vitamin E 40.5 8.1 4.198.4 7.2 7.2 Aqueous TPGS 6.4 1.28 Phase Pluronic 5.0 1.0 F127 Water395.25 79.05 41.0 82.0 79.5 79.5 Total 500.0 100.0 52.2 104.4 102.6102.6

Example 35

[0196] Compositions of various self-emulsifying emulsions useful in thisinvention are shown in Table 7. TABLE 7 Self-Emulsifying EmulsionsComposition of SEFP-1 SEFP-2 Self-Emulsifying Weight Weight WeightWeight Emulsions (g) % (g) % Paclitaxel 0.255 5.11 0.258 5.14 Vitamin E1.989 19.88 2.242 44.70 TPGS 0.992 19.99 0.765 15.25 PEG 400 1.502 30.110.999 19.92 Pluronic F127 0.250 5.01 Solutol HS15 0.752 14.99 Total4.988 100.00 5.016 100.00

[0197] The emulsions described in Table 7 were synthesized as follows.

[0198] SEFP-1

[0199] Paclitaxel and PEG 400 were heated together at 60-67° C. andstirred until the drug was dissolved in PEG (15 min). Then TPGS andPluronic F127 were added and stirred at 70° C. for 10-15 min to dissolvethe surfactant. Finally, Vitamin E (α-tocopherol) was added and mixedfor 5-10 min at 55° C. until the mixture was clear and homogeneous. Upondilution with an aqueous phase a fine emulsion can be obtained.

[0200] SEFP-2

[0201] Paclitaxel and PEG 400 were first stirred at 65-75° C. for 45 minthere TPGS was added and stirring was continued for another 30 min tocompletely dissolved all three components and produced a clear solution.Finally Solutol HS-15 and Vitamin E were added and mixed for about 5 minat 55° C. to obtain a clear homogeneous liquid. Upon dilution with anaqueous phase a fine emulsion can be obtained.

Example 36

[0202] Additional compositions of self-emulsifying emulsions ofpaclitaxel are shown in Table 8. TABLE 8 Self-Emulsifying EmulsionsComposition of Self- SEFP-3 SEFP-4 Emulsifying Weight Weight WeightWeight Emusions (g) (%) (g) (%) Paclitaxel 0.10 2 0.05 1 α-Tocopherol1.40 28 0.50 10 TPGS 1.00 20 0.95 19 PEG400 1.00 20 1.00 20 SolutolHS-15 1.50 30 2.50 50 Total 5.00 100 5.00 100

[0203] SEFP-3 and SEFP-4 were prepared by first dissolving paclitaxel insolutol HS-15+PEG 400 by low shear mixing at 60-70° C. (<30 min), TPGSand α-tocopherol were then added and briefly mixed to form a clearsolution (TPGS solidification can be observed at room temperature butremains a clear liquid at 37° C.

[0204] The particle size of the emulsions upon dilution of SEFP-3 andSEFP-4 was determined as follows: 0.2 ml of SEFP-3 or SEFP-4 was dilutedin 100 ml of Phosphate-buffered Saline at 37° C. by low shear mixingwith a stir bar for 5 minutes. An emulsion was quickly formed, theparticle size of which was measured by the Malvern Mastersizer. Thevolume mean diameter of SEFP-3 and SEFP-4 was found to be, 2.49 and 1.55μm, respectively.

[0205] For an efficient self-emulsified system the mean droplet diameterof the resulting emulsion should be less than 10 μm and preferably lessthan 5 μm.

Example 37 Paclitaxel Emulsions Incorporating a Pegylated Phospholipid

[0206] DMPE-PEG₂₀₀₀ (Dimyristoyl Phosphatidyl Ethanolamine-PolyethyleneGlycol 2000) incorporating emulsions were prepared (Table 9).Paclitaxel, when present, was first dissolved in PEG 400 by low shearmixing at 75° C. The other ingredients were added and briefly mixed(after melting TPGS, and in the case of DMPEG-2, the P 407) to form aclear solution. A vacuum was applied to degas the oil phase prior toemulsification, and the oil phase was brought to 45° C. Water was boiledfor 15 minutes to degas, then brought to 45° C. also. The two phaseswere mixed at 45° C. at low to medium shear to form a pre-emulsion. Forformulations DMPE-PEG-P2, DMPE-PEG-P3 and DMPE-PEGP-4, this wasaccomplished by simply adding the warm water to the oil phase andswirling by hand with sonication. The pre-emulsion for DMPE-PEG2 wasprepared by pouring oil phase into water while stirring with alaboratory mixing motor. Pre-emulsions were immediately homogenized inthe Avestin C5 homogenizer at 20-22K psi peak stroke pressure to producefine emulsions with a mean droplet diameters and 99% cumulativedistributions of less than 200 nm. TABLE 9 Paclitaxel EmulsionsIncorporating a Pegylated Phospholipid DMPE- DMPE- DMPE- DMPE- PEG-1PEG-2 PEG-3 PEG-4 (g) (%) (g) (%) (g) (%) (g) (%) Paclitaxel 0.53 1.10.96 1.0 PEG 400 3.07 6.1 5.77 5.8 1.8 6.0 1.84 6.1 TPGS 2.59 5.1 4.624.7 1.51 5.0 1.22 4.1 DMPE-PEG₂₀₀₀ 0.53 1.1 0.20 0.2 0.30 1.0 0.62 2.1Poloxamer 407 0.96 1.0 Vitamin E 4.11 8.2 7.71 7.8 2.42 8.1 2.14 7.2Water 39.50 78.5 79.00 79.6 24.0 79.9 24.1 80.5 Total 50.33 100.0 99.23100.0 30.03 100.0 29.92 100

Example 37 Efficacy Data

[0207] Formulation D (Table 6) was evaluated for efficacy against B16melanoma in mice as described in Examples 18 and 19 and the data issummarized in FIG. 5. Comparative efficacy data is presented in Table10. TABLE 10 Comparative Efficacy in B16 Melanoma Tumor Model: Taxol ®vs SONUS Paclitaxel Emulsion “QW 8184” Total % % Dosage Dose Mortal- T/CLog Test (mg/kg/ Schedule (mg/ Median Tumor Weight on Day ity (by Day T− C cell Article day) (days) kg) 1 4 7 10 13 17 day 17) 13 (days) killSaline 80 q4dx5 — 8 245 1271 1800 2916 14114 60 — — — equiv. 0 Taxol ®20 qdx5 100 6 123 331 2 2192 4901 20 75 5 0.9 9 Formu- 60 q3dx5 300 1106 221 234 400 400 60 14 13 2.3 lation 0 D 8

[0208] Consistent with the data in Table 4, efficacy assessment by tumorgrowth inhibition, tumor growth delay and log cell kill indicatesignificant improvement with Formulation D over Taxol®.

Example 38 Physical Stability Data

[0209] The physical stability of formulation D was assessed by potentialparticle size changes upon storage and the data is shown in Table 10.TABLE 11 Physical Stability of Formulation D Volume-weighted ParticleSize (nm) Storage Day Mean Droplet Distribution 99% of (2-8° C.)Diameter the Particles less than 2 71.3 154.6 3 69.3 151.8 10 67.7 151.615 69.8 150.8 28 66.3 152.3 30 66.9 150.3

[0210] Particle size was measured using the Nicomp 370 sub-micronparticle size analyzer. As can be seen from the data in Table 11 nosignificant changes were observed in either the mean droplet diameter orthe 99% cumulative distribution of the particles. The latter parameteris often used as an indicator of particle aggregation and growth. Inaddition, no precipitation or other gross changes were observed duringstorage. Long term stability is ongoing.

Example 39 Chemical Stability

[0211] The chemical stability of formation D (Table 6) was monitored byHPLC using the procedures of Example 5 and the data is shown in Table12. HPLC is utilized to quantitate the concentration of paclitaxel anddegradants. In Table 12, drug concentration is equivalent to drugpotency. TABLE 12 Chemical Stability of Formulation D Storage Day DrugConcentration (2-8° C.) (mg/ml) 0 9.53 10 9.54 19 9.39 32 9.54

[0212] It is evident from this data that the drug potency in formulationD remains unchanged under these storage conditions.

[0213] In addition, no degradation of the drug was observed during thisstorage time.

Example 40 Emulsions Containing PEG 300 or NMP

[0214] α-Tocopherol emulsions containing PEG 300 or NMP(N-Methyl-2-pyrrolidone) and incorporating 10 mg/ml paclitaxel are shownin Table 13. TABLE 13 PEG 300 NMP Weight Weight Weight Weight Component(g) % (g) % Paclitaxel 0.05 1.0 0.05 1.0 PEG 300 0.32 6.2 NMP(N-Methyl-2- 0.18 3.6 pyrrolidone) TPGS 0.25 4.9 0.25 5.0 Poloxamer 0.050.9 0.05 1.0 407 Vitamin E 0.40 7.9 0.43 8.7 Water 4.00 78.9 4.00 80.7Total 5.07 100.0 4.96 100.0

[0215] In both cases, paclitaxel was first dissolved in the solvent (PEG300 or NMP) with low shear mixing. Heating to 60° C. was used with thePEG 300 to speed the dissolution while with the NMP formulation a fewminutes at room temperature was sufficient to dissolve the drug. Theremaining ingredients (except water) were then added and the mixtureswere heated to 60-65° C. with low shear mixing to melt the solidsurfactant and produce homogeneous, clear solutions. The solutions werebrought to 45° C., then 45° C. water was added to them. The resultingmixtures were processed under medium shear to produce a thick, whitecrude emulsion, very similar in appearance to the pre-emulsion offormulation D (Table 6). These emulsions can further be homogenized athigh pressure to produce fine emulsions.

Example 41 Large Scale Preparation of Formulation D (QW 8184)

[0216] Using procedures analogous to those described in previousexamples, formulation D (Table 6) was manufactured at a large scale in2×2L sub-lots having the following composition (the shaded arearepresents the oil and aqueous phase content of the emulsion): TABLE 14Sub-Lot 1 Sub-Lot 2 Amount in Amount in Oil Phase Weight Oil PhaseWeight Component (g) (%) (g) (%) Paclitaxel 21 1.01 21 1.01 PEG400 123.65.96 123.6 5.92 α-Tocopherol 164.8 7.94 164.8 7.89 TPGS 103 4.97 1034.93 Poloxamer 407 20.6 0.99 20.6 0.99 Oil Phase 433 20.97 413.2 20.73Total

[0217] For the preparation of the pre-emulsion, 416.8 g of the oil phaseof sub-lot 1 and 413.2 g of the oil phase of sub-lot 2 were mixed with1580 g of water for injection (5 min at 46° C.). Upon homogenizationfine emulsions were produced having a mean droplet diameter of about 70nm, that is, very similar to that of formulation D at the small scale(Table 10). This scaled formulation was further sterilized by filtrationthrough a 0.2 micron filter.

Example 42 Hemolytic Activity Evaluation of a Drug-Free Emulsion

[0218] A large scale (2.5 L) of formulation D in the absence ofpaclitaxel was prepared as described in Example 41 having the followingcomposition. TABLE 15 Amount in Oil Phase Component (g) Weight % PEG400154.5 5/97 α-Tocopherol 206 7.96 TPGS 128.8 4.97 Poloxamer 407 25.8 1.00Oil Phase 515.1 19.89 Total

[0219] For the preparation of the pre-emulsion, 496.7 g of the oil phasewere mixed with 2000 g of water for injection (5 min at 46° C.). Uponhomogenization and filter sterilization this formulation was evaluatedfor gross hemolytic reaction with human blood using the followingprocedure:

[0220] Volunteer healthy blood was collected with heparin by Vacutainerstick. The plasma was initially straw colored and negative forhemolysis. Drops of whole blood and the drug-free emulsion were broughttogether under coverslip and observed microscopically for severalminutes. During contact, red blood cells (RBCs) remained normocytic. Noobvious aggregation of the emulsion particles was noted. No grosschanges in platelet or WBC morphology were noted. Then, in test tubes,whole blood and vehicle were mixed 1:1 and 5:1, v/v. As a control, wholeblood was mixed with saline for injection 1:1. All mixtures wereincubated at 37° C. and examined at 10 and 30 min. Supernatants in allthree tubes were straw colored and clear. It can be concluded form thisstudy that there is no immediate gross hemolytic reaction between theemulsion vehicle and blood. This suggests that the morphology of the redcell membranes is not perturbed by the surfactants present in theemulsion, in contrast to several reports in the literature onsurfactant-induced hemolysis of RBC.

Example 43 Physical Stability Data

[0221] Table 16 shows long-term stability of the scaled up formulationof Example 41 upon a 9-month storage at 4° C. or 25° C. It is evidentthat at least during this storage time, both the mean droplet diameterand the 99% cumulative distribution did not significantly changed fromtheir initial values of about 65 and 150 nm, respectively, and theemulsion remains within specifications. TABLE 16 Physical Stability ofQW8184 99% Cumulative Storage Mean Droplet Diameter, Distribution, nmTime nm (mean ± sd) (mean ± sd) (months) 4° C. 25° C. 4° C. 25° C. 0.064 ± 0.8 63 ± 2.1 150 ± 0.7 150 ± 0.7 0.5 67 ± 2.9 63 ± 2.5 152 ± 2.8149 ± 3.6 1.1 64 ± 2.5 65 ± 2.5 149 ± 2.0 152 ± 2.1 3.1 66 ± 1.2 62 ±2.0 150 ± 1.2 148 ± 2.5 6.1 63 ± 1.2 64 ± 3.1 150 ± 1.5 152 ± 4.0 9.2 64± 2.1 62 ± 1.0 152 ± 2.1 153 ± 0.7

Example 44 Chemical Stability

[0222] A 9-month chemical stability data of the scaled up formulation ofExample 41 in terms of paclitaxel potency and levels of known degradantsare shown in Tables 17 and 18. As can be seen from these results, therewere no significant changes in either the drug potency or the levels ofknown degradants and the product remains within specifications at bothstorage temperatures. TABLE 17 Paclitaxel Potency and Degradants at 4°C. Degradants Paclitaxel (%, mean ± sd, Storage Potency n = 3) Time mean± sd, n = 3 7-Epi- 10-Deacetyl- (months) (mg/ML) paclitaxel Baccatin-3paclitaxel 0.0 8.22 ± 0.64 0.17 ± 0.01 0.12 ± 0.01 0.15 ± 0.01 0.5 9.48± 0.08 0.32 ± 0.05 0.15 ± 0.00 0.16 ± 0.00 1.1 8.79 ± 0.53 0.31 ± 0.030.17 ± 0.00 0.17 ± 0.00 3.1 9.50 ± 0.07 0.61 ± 0.03 0.20 ± 0.00 0.20 ±0.00 6.1 9.27 ± 0.17 0.28 ± 0.02 0.17 ± 0.01 0.18 ± 0.02 9.2 9.21 ± 0.120.36 ± 0.02 0.17 ± 0.00 0.18 ± 0.01

[0223] TABLE 18 Paclitaxel Potency and Degradants at 25° C. DegradantsPaclitaxel (%, mean ± sd, Storage Potency n = 3) Time mean ± sd, n = 37-Epi- 10-Deacetyl- (months) (mg/ML) paclitaxel Baccatin-3 paclitaxel0.0 8.22 ± 0.64 0.17 ± 0.01 0.12 ± 0.01 0.15 ± 0.01 0.5 9.10 ± 0.65 0.33± 0.00 0.17 ± 0.00 0.17 ± 0.01 1.1 8.06 ± 0.75 0.32 ± 0.04 0.17 ± 0.000.17 ± 0.01 3.1 9.19 ± 0.79 0.65 ± 0.05 0.22 ± 0.00 0.22 ± 0.00 6.1 9.11± 0.71 0.33 ± 0.02 0.16 ± 0.02 0.15 ± 0.03 9.2 9.02 ± 0.68 0.36 ± 0.020.18 ± 0.01 0.18 ± 0.01

Example 45 Efficacy Evaluation

[0224] The formulation of Example 41 was evaluated for efficacy againstB16 melanoma as described in Examples 18, 19 and 37 and the results aresummarized in Table 19. TABLE 19 Antitumor Activity of QW8184 vs Taxol ®in the B16 Melanoma Model Dose Survival Test mg/kg Schedule (mean ± SD)% T/C^(a) % TGI^(b) T − C^(c) Log Cell Article n = 8 Days days day 20day 20 days Kill^(d) Saline Control q3dx5 17 ± 2 — — — — Vehicle Controlq3dx5 20 ± 1 93 3 3 — Taxol ® 20 q3dx5 19 ± 5 77 23 3 0.5 QW8184 20q3dx5 28 ± 7 11 89 10 1.8 QW8184 40 q3dx5 33 ± 5 0 100 17 3.0

[0225] By all end points of efficacy, QW8184 exhibited superiorantitumor activity in mice at doses that included or well exceeded theMTD of Taxol® but which were well tolerated. Such effects have not beenreported with previous injectable emulsions of paclitaxel. MTD is themaximum tolerated dose that is determined from acute toxicity studies.

Example 46 Efficacy Evaluation

[0226] The antitumor activity of QW8184 (Example 41), against the humanovarian tumor xenograft IGROV-1 using the marketed product Taxol® as areference formulation. Nude mice were implanted subcutaneously by trocarwith fragments of IGROV-1 human ovarian carcinomas harvested fromsubcutaneously growing tumors in nude mice hosts. When tumors wereapproximately 5×5 mm in size, the animals were paired matched intotreatment and control groups contained 9 ear-tagged tumor-bearing miceper group. QW8184 was administered i.v. on a q3dx5, q4dx5, and qdx5schedule at 20, 40 and 60 mg/kg. Taxol® was administered i.v. on thesame schedules at 20 mg/kg its maximum tolerated dose. Mice were weighedtwice weekly, and tumor measurements were taken by calipers starting Day1 and converted to mg tumor weight. The experiment was terminated whenthe control tumors reached approximately 1 gr and tumors were excisedand weighed and the mean tumor weight per group was calculated. The datais summarized in Table 20. TABLE 20 Antitumor Activity of QW8184 vsTaxol ® in the IGROV-1 Human Ovarian Tumor Xenograft Mice with DoseFinal Tumor Wt Complete Group Schedule (mg/kg) (Mean ± SEM, mg) % TGIShrinkage Saline q3dx5 control  874.8 ± 178.6 — 0 QW8184 q3dx5 vehicle839.9 ± 80.4 4.4 0 QW8184 q3dx5 20 115.9 ± 39.1 93.4 2 QW8184 q3dx5 40 0.1 ± 0.1 — 8 QW8184 q3dx5 60  0.0 ± 0.0 — 7 QW8184 q4dx5 20  69.2 ±28.4 99.9 3 QW8184 q4dx5 40  0.0 ± 0.0 — 9 QW8184 q4dx5 60  4.9 ± 4.9 —8 QW8184 qdx5 20 158.2 ± 56.7 88.7 3 Taxol ® q3dx5 20  22.3 ± 14.2 — 3Taxol ® q4dx5 20  24.0 ± 11.5 — 3 Taxol ® qdx5 20 16.7 ± 9.6 — 2

[0227] Administration of QW8184 at 20, 40 and 60 mg/kg on a q3dx5 orq4dx5 schedule resulted in nearly 100% tumor growth inhibition at alldoses with 2, 8, and 7 and 3, 9, and 8 complete tumor responses,respectively. In comparison, administration of Taxol® resulted in 3complete tumor responses on both schedules. On a qdx5 schedule, theantitumor activities of QW8184 and Taxol® were similar. QW8184, however,was better tolerated with no toxic deaths whereas six toxic deaths werenoted with Taxol®. QW8184 was highly active against the IGROV-1 humanovarian xenograft model in a dose-dependent fashion, regardless of thedosing schedule and it was better tolerated than Taxol®.

Example 47 Pharmacokinetic Study

[0228] The pharmacokinetics of the formulation of Example 41 (QW8184),in the rat upon a single 10 mg/kg i.v. administration was determinedusing Taxol® as a reference formulation. The drug was administered i.v.to male or female rats either as a 3-hr infusion (Taxol®) or as a bolusdose (QW8184). Blood samples were collected from 0-72 hrs after doseadministration, plasma was prepared by centrifugation and analyzed forpaclitaxel concentration using a high performance liquid chromatography(HPLC) method with LC/MS/MS detection. Pharmacokinetic analysis wasperformed on the mean composite plasma concentration-time profiles usinga model independent method. The derived pharmacokinetic parameters areshown in Table 21. The pharmacokinetic parameters determined were asfollows:

[0229] T_(max): time required to reach peak plasma levels (C_(max))

[0230] C_(max): peak plasma concentration of the drug

[0231] AUC_(O-t): non-extrapolated area under the plasmaconcentration-time curve from time zero to time t which is the end ofthe plasma sample collection

[0232] AUC_(0-∞): extrapolated area under the plasma concentration-timecurve from time zero to infinite TABLE 21 Derived PharmacokineticParameters of Paclitaxel Following Intravenous Administration of QW8184or Taxol ® in Rats at 10 mg/kg (70 mg/m²) Pharmaco- kinetic QW8184Taxol ® Parameter Male Female Male Female T_(max) (hr) 0.083 0.083 3 3C_(max) (ng/mL) 58950 53900 5867 7227 AUC_(0-t) 35504 32761 18138 22701(ng · hr/mL) AUC_(0-∞) 35551 32829 18347 23002 (ng · hr/mL) K_(e) (hr⁻¹)0.0940 0.1375 0.1283 0.0754 T_(½) (hr) 7.38 5.04 5.40 9.20 V_(d) (L/kg)2.99 2.22 4.25 5.77 CL (L/ 0.281 0.305 0.545 0.435 hr/kg) V_(SS) (L/kg)0.228 0.242 1.44 1.09

[0233] Both the C_(max) and AUC_(0→∞) values following the i.v. bolusadministration of QW8184 were significantly higher than thecorresponding values following the i.v. infusion of Taxol®. The terminalT_(½) of paclitaxel in plasma were similar for the two treatments.Tissue binding was more extensive with Taxol® than QW8184 as indicatedfrom differences in the volume of distribution at steady state (Vss). Nosignificant differences in the pharmacokinetic parameters of paclitaxelwere observed between male and female animals.

We claim:
 1. A method of making an emulsion, comprising: combining achemotherapeutic agent selected from taxoids, taxines,microtubule-targetting agents, and taxanes; a co-solvent; one or moresurfactants, tocopherol and an aqueous phase to form an emulsion.
 2. Themethod of claim 1 wherein said co-solvent is selected fromdimethylsulfoxide, dimethylamide, ethylene glycol, propylene glycol,glycerol, sorbitol, mannitol, polyethylene glycol,N-methyl-2-pyrrolidone, polyvinyl-pyrrolidone or mixtures thereof. 3.The method of claim 1 wherein said surfactant is an α-tocopherolderivative.
 4. The method of claim 2 wherein said polyethylene glycolhas a molecular weight between 100 to 10,000.
 5. The method of claim 2wherein said polyethylene glycol has a molecular weight between 200 to600.
 6. The method of claim 2 wherein said co-solvent isN-methyl-2-pyrrolidone.
 7. The method of claim 3 wherein saidα-tocopherol derivative is an ester or an ether of α-tocopherol andpolyethylene glycol.
 8. The method of claim 3 wherein said α-tocopherolderivative is D-α-tocopherol-polyethylene glycol 1000 succinate.
 9. Themethod of claim 10 wherein said second surfactant is selected from thegroup consisting of anionic, cationic, nonionic and zwitterionicsurfactants.
 10. The method of claim 10 wherein said second surfactantis selected from the group consisting of poloxypropylene-polyoxyethyleneglycol nonionic block polymers, ascorbyl-6-palmitate, stearylamine andsucrose fatty esters.
 11. The method of claim 1 wherein said taxane ispaclitaxel.
 12. A method of making an emulsion, comprising: a)dissolving one or more chemotherapeutic agents selected from taxoids,taxines, microtubule-targetting agents and taxanes in a co-solvent isselected from dimethylsulfoxide, dimethylamide, ethylene glycol,propylene glycol, glycerol, sorbitol, mannitol, polyethylene glycol,N-methyl-2-pyrrolidone, polyvinyl-pyrrolidone or mixtures thereofsolvent to form a chemotherapeutic agent solution; b) adding one or moresurfactants to said chemotherapeutic agent solution to form an oilsolution and c) adding α-tocopherol to said oil solution to form anα-tocopherol-chemotherapeutic agent solution.
 13. The method of claim 13including the additional step d) blending saidα-tocopherol-chemotherapeutic agent solution with an aqueous phase toform a pre-emulsion.
 14. The method of claim 14 including the additionalstep e) homogenizing said pre-emulsion to form a fine emulsion.
 15. Themethod of claim 13 wherein said solvent is polyethylene glycol.
 16. Themethod of claim 15 wherein the molecular weight of said polyethyleneglycol is between 300 and 500.